Genetic Engineering Subtilisin To Selenium-containing Antioxidant Enzyme With High GPx Activity

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 harmacological use of the naturally occurring enzymes.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. On the groundwork of the previous work, we rationally redesigned of the active site of enzymes with combination of computer-aided molecular simulation and rebuilding the catalytic site to gain the novel selenoenzyme with enhanced GPx activity.1. Engineering subtilisin into efficient GPx mimic.The serine protease subtilisin is an important industrial enzyme as well as a model for understanding the enormous rate enhancements affected by enzymes. For these reasons along with the timely cloning of the gene, ease of expression and purification and availability of atomic resolution structures, subtilisin became a model system for protein engineering studies in the 1980s. In 1990, Hilvert and coworkers transformed subtilisin Carlsberg (from Bacillus licheniformis) into seleno-subtilisin by a chemical modification method. However, chemical modification is incapable of specifically targeting amino acid residues in the active site, and in some case, other hydroxyl groups in the protein are inevitably converted into selenols, which will interfere with the further structure function studies of the redesigned selenoenzyme. In fact, site-directed mutagenesis has become an indispensable tool for altering activities and selectivities of enzymes. In view of this, genetic engineering should provide a better and more suitable alternative to incorporate Sec into the defined protein's active site. Based on an auxotrophic expression system, B?ck et al. have performed the study of combining diselenocysteine with thioredoxin. Using a previously established procedure, cysteine residue in the active site of the phosphorylating glyceraldehydes 3-phosphate dehydrogenase (GAPDH) was converted to a Sec residue and also revealed a novel peroxidase activity. More recently, our group has performed the transformation of glutathione transferase (Lucilia cuprina LuGST1-1) into selenoenzyme (seleno-LuGST1-1) by means of the auxotrophic expression. However, very low yield of 1~4 mg selenoprotein per liter medium in soluble expression can be obtained in these expression systems. Since the element of selenium has some unstable properties, no research group tried to gain the proteins from inclusion bodies. Considering these reasons, we tried to extract proteins from inclusion bodies produced in the auxotrophic expression system in Escherichia coli.In this investigation, we employed the wild-type subtilisin E (from Bacillus subtilis) as a template and mutated the serine221 in the active site of the enzyme to cysteine, and then biosynthetically substituted cysteine to selenocysteine in a cysteine auxotrophic expression system. The abundant protein was purified to homogeneity in the presence of 5M urea. Upon in vitro renaturation of the denatured protein, the efficiency of protein refolding was high up to 60~80 %. Especially, we obtained 80~100 mg activated seleno-subtilisin E per liter medium in the auxotrophic expression system for the first time. This work will become the groundwork for the future study aimed at correlating enzyme structure with chemical function by further rebuilding the active centre of seleno-subtilisin.2. Rational redesign of the active site of seleno-subtilisin with enhanced GPx activity.Enzyme redesign for optimizing the catalytic properties is a most promising and challenging topic with great current interest. With the help of advantageous tools of enzyme engineering such as site-directed mutagenesis and three-dimensional structure prediction by the computer-aided molecular simulation, the redesign of active sites of native enzymes gains importance for altering the enzyme activity and for obtaining new enzyme activity.One of the most significant challenges for rebuilding and optimizing enzyme properties is rational design and redesign of the active sites of enzymes. For doing so, the combination of the strategy of enzyme engineering with the computer-aided design becomes an indispensable approach.In order to broaden substrate specificities and improve trace activity of selenosubtilisin, we redesigned the active site of selenosubtilisin by transferring the catalytically essential Sec to the edge of the substrate-binding pocket of the enzyme. From the structure prediction for the binding of the substrate ArSH in the active site of subtilisin by computer-aided modeling, we found that the minimum energy state made the thiol group of ArSH turn outward to approach the residue Ser63, which was located at the border of the binding pocket in selenosubtilisin. Based on the interaction of substrate ArSH and the related amino acid residues in the active site, the catalytic essential Sec should be changed from position 221 to 63. In this case, ArSH had a slight shift from the bottom to the outside, the position of ArSH was moved outward about 3? (from 9.23 to 6.22 ?) to the border of the pocket, however, the orientation of the thiol group of ArSH changes obviously, it turns from inward direction of the active-site cleft to outward. The alteration of the essential catalytic Sec from the bottom position 221 to the 63 of the edge of the binding pocket should display evident advantage for improving the catalytic activity. In seleno221-subtilisin, the selenium side chain is buried in such a deep pocket and is presumably less accessible for its substrate hydroperoxides. After this alteration the mode of the substrate ArSH binding to the enzyme changed accompanyingly, the optimized redesign led to remarkable improvement of seleno-subtilisin catalytic efficiency.The novel seleno63-subtilisin E showed a prominent enhancement of glutathione peroxidase (GPx)-like activity, for example, the GPx activity of seleno63-subtilisin for the reduction of cumene hydroperoxide (CUOOH) by 3-carboxy-4-Nitroben-zenethethiol (ArSH) was high as 2570μmol·min-1·μmol-1 at physiological condition and approached the level of some naturally occurring GPxs (e.g. 5780μmol·min-1·μmol-1 from rabbit liver). The second-order rate constant kmax/KmArSH of seleno63-subtilisin catalysis for the reduction of t-BuOOH by ArSH was found to be in the same order of magnitude as that of the natural GPx, and the kmax/Km t-BuOOH showed two to three orders of magnitude increasing than that of the original seleno221-subtilisin E. Compared with diphenyl diselenide (PhSeSePh), a well studied GPx mimic, this engineered selenoenzyme exhibited striking active enhancement of 3700, 000-fold. More importantly, seleno63-subtilisin E displays not only high GPx-like activity but also remains efficient hydrolase activity.