Construction Of High Efficient Antioxidant Enzyme By Genetic Engineering

Enzymes are proteins with special function, which can catalyze certain chemistry reaction specificly in mild conditions. Because of the unique supramolecular interaction between enzyme and its substrate, it becomes the first choice when studying the bionic system. Artificial simulation of the molecule recognition and catalysis of enzyme is significant for understanding its intrinsic biological evolution process and relationship between the structure and function. Design and synthesis of simulative enzyme with highly efficient stereoselectivities and thorough comprehension of the catalysis mechanism is a goal the scientists are pursuing all along.Organism obtain energy by reducing O₂ in natural physiological condition, O₂^-·,·OH and H₂O₂ engendered in the metabolism are irreversible production, they are called Reactive Oxygen Species(ROS) together with the unstable intermediate of lipid peroxide. Under normal conditions, there is a balance between the production and destruction of ROS. In certain pathogenic states, the balance is broken and the accumulated ROS harm various biomacromolecules including RNA, DNA, protein, sugars and lipids, which lead to cell dysfunction. Therefore these result in ROS-mediated diseases include Alzheimer s disease, cardiac muscle shock, atherosclerosis, immuno-deficiency, radiation damage, emphysema, cataract and other age-related diseases. In order to eliminate the excess ROS, the organism sets up a series of defensive system, including enzymatic and non-enzymatic antioxidant systems. The enzymatic antioxidant system consists of glutathione peroxidase(GPx), catalase(CAT) and superoxide dismutase(SOD). GPx is the important member of the enzymatic antioxidant system, which can catalyze the decomposition of H₂O₂ using GSH as the substrate. It can also interdict the second-class reaction of the radical which is produced by lipid peroxide. Thus, GPx could be a promising antioxidant drug. However, the disadvantage such as difficult heterogenous expression, solution instability, short half-lives and proteolytic digestion limited the pharmacological application of the naturally occurring enzymes. Enormous efforts have been made to simulate the highly efficient functions of GPx. Not only can it be important for elucidating the relationship between structure and function of enzyme, but also provide possibility for preparing efficient preventive and curative drug for ROS-mediated diseases.The 21st amino acid selenocysteine (SeCys) is the catalytic center of GPx. Its triplet coden UGA is usually a stop coden, and needs a special modulation mechanism when incorporated into protein. Therefore it is difficult to express SeCys by traditional recombinant DNA technology. Heretofore, most of the GPx mimics were prepared with chemical methods. The small molecules mimics thereinto generally demonstrated relatively low activity, due to the neglect of the substrate binding. While the activity of the macromolecules mimics (such as antibody enzyme and bio-imprinting enzyme) which were based on the substrate binding have been improved a lot. But the limitation of the methods used in the preparation made their structure difficult to characterize clearly, which blocked the further studies of catalytic mechanism and structure-function relationship.The E.coli. auxotrophic expression system is a new developed method recently, which is based on the following assumption: the aa-specific tRNA is aminoacylated with amino acid analogue by aa-tRNA ligase when the amino acid is omitted. The E.coli. cysteine auxotrophic expression system employed in this dissertation just take the point that the E.coli. strains of cysteine auxotrophic can not synthesize cysteine itself, so it can make full use of the selenocysteine or tellurocysteine abundant in the culture medium to express target protein. The advantage is the high selenocysteine or tellurocysteine incorporation rate. What is more, it will not change the conformation of the protein, thus will keep its character.In enzyme engineering, most of the novel function of enzyme were acquired by redesign of existing protein scaffolds: namely, the rational reconstruction of the enzyme structure to achieve the anticipated functional transformation. This enlightens us the redesign based on existing protein scaffolds to construct high efficient antioxidant enzyme. Therefore, we take the Lucilia cuprina glutathione transferases (LuGST1-1) as the protein scaffold, and make full use of the cooperation of substrate binding and catalysis to construct GPx mimic. Meanwhile, we study the structure-function relationship of the mimic to explore the principle of catalysis.1. Construction of GPx mimic by using telluro-LuGST1-1GSTs are important phase II detoxification enzymes found mainly in the cytosol. And both GST and GPx belong to thioredoxin superfamily due to the thioredoxin fold in their structure. They have the similar glutathione-binding domain; the orientation of the catalytic residues are the same though the functions are different. In addition, some class of GST were reported to have GPx activity towards organic peroxide. Tellurium follows selenium in group VI, so they have similar redox characteristic, and tellurol reduces peroxide more easily than selenol. The cyclic voltammograms approved that tellurocysteine (TeCys) has an intrinsically lower redox potential (-850 mV versus Ag/AgCl) than that of SeCys (the catalytic center of GPx; -640 mV versus Ag/AgCl). As a result, proteins containing TeCys residues can participate in unique and biologically fundamental redox reactions. The reduction of tellurocystine by GSH was confirmed by HPLC and UV/visible spectrometry. Though TeCys is toxic to cells, the aminoacylation of tRNA with TeCys clearly showed that CysRS can aminoacylate tRNAcys with TeCys effectively. All the above provided solid theoretical evidence for our idea: employing E.coli. cysteine auxotrophic expression system to introduce TeCys into the LuGST1-1 protein scaffold as catalytic center, making full use of the natural occurring GSH binding site, to get a more efficient GPx mimic than its SeCys analogue. The so-made telluro-LuGST1-1 is the first TeCys incorporated protein produced by genetic engineering. We improved the expression condition due to the toxicity of tellurium, the principle is: reducing the cellular toxicity of TeCys and making it fully used. The telluro-LuGST1-1 was well characterized.The GPx activity of the telluro-LuGST1-1 was improved further, its GPx activity towards GSH was at the same level as that of some natural GPxs. Its enzymology character was close to natural GPx, and exhibited good stability during the catalysis and preservation. The steady kinetics indicated a sequential mechanism. The kinetic constants k_max/K_H2O2 for the telluro-LuGST1-1 was one magnitude lower than the natural GPx, while the k_max/K_GSH was even the same level as the natural GPx. Via structure comparison, we concluded that the high efficiency of telluro-LuGST1-1 were from the following aspects. The striking thioredoxin fold structural similarity between the specific glutathione-binding domain folds in LuGST1-1 and GPx; the similarity in the orientation of their catalytic center with vicinalβαβmotif; its catalytic center TeCys has an intrinsically lower redox potential than that of SeCys (the catalytic center of GPx). The results indicated that the specific substrate binding site and the exact orientation of the catalytic center are important for acquiring high efficient enzyme mimics.Successful engineering of LuGST1-1 into a high efficient GPx mimic provided a new proof for the previous assumption that both GPx and GST were evolved from a common ancestor. Meanwhile, it supports that the dominant factor in the evolution of new enzymatic function is chemistry selectivity instead of binding specificity. The viable general method for the synthesis of telluroenzymes with GPx activity can be extended to more proteins models. It will continue to open new avenues for further investigation of tellurium chemistry in proteins, not only for structure-function studies, but also for medical applications. 2. Construction of GPx mimic by using seleno-LuGST1-1GST can catalyze the conjugation of electrophilic aryl substrates to glutathione (GSH), and the most mentioned is with 1-chloro-2,4-dinitrobenzene (CDNB). SeCys and TeCys had been introduced into subtilisin by chemical modification to achieve GPx activity using 3-carboxy-4-nitrobenzenethiol (CNBSH) as substrate. But the subtilisin is a hydrolase, and the structure of its natural substrate is different from CNBSH, so the GPx activity of selenosubtilisin and tellurosubtilisin towards CNBSH could be improved.With the instruction of computer simulation and former study, Tyr113 was selected as reconstruction target, which locates in the natural substrate CDNB binding site of LuGST1-1 and has no dominant effect on the protein structure. Similarly, we used E.coli. cysteine auxotrophic expression system to introduce SeCys into the LuGST1-1 protein scaffold as catalytic center. We explored the expression condition to obtain single objective protein which was beneficial for further purification, and renatured the inclusion body employed the gradient urea Sephadex column refolding method. The circular dichroism proved the successful renaturation. Three mutants were designed and the GPx activities were determined to investigate the role the residues played. The results confirmed the replacement of Tyr113 with SeCys was crucial for catalysis: not only can make use of the similarity of the CNBSH with CDNB (the natural substrate of LuGST1-1), but also can stabilize the anion. So the introduction of SeCys to this site as catalytic center can exert efficiency better. Seleno-LuGST1-1 demonstrated the highest activity towards CUOOH among the three peroxides because of the stereo-hindrance. Double-reciprocal plots of the initial velocity versus substrate concentration yielded a series of intersecting linear plots, which indicated a sequential mechanism. The results indicated once again that the substrate binding and the orientation of the catalytic center are important for acquiring high efficient enzyme mimics.