Computational Studies On Structures, Functions And Modulations Of Several Oxidoreductases

The level of oxidant and antioxidant systems of biological organisms is usually balanced in a constant redox state, thus maintaining the stability of internal environment. In the setting of oxidation/antioxidation imbalance where large amounts of highly reactive molecules such as reactive oxygen species (ROS) are generated, biological system tends to be in an oxidized state. When the production of ROS is out of the control of biological organisms, oxidative damage will be caused. The phenomenon is refered to oxidative stress (OS). OS is involved in many diseases such as atherosclerosis, diabetes, cardiovascular disease, heart failure, tumor, Parkinson’s disease, and Alzheimer’s diseases and so on.To counteract oxidative damage resulting from OS, a set of perfect oxidation antioxidation systems has been developed in biological organisms. The systems include NADH/NAD+, NADP+/NADPH, and a series of proteins such as thioredoxin family and transcriptional repressor Rex which responds to the redox state.In this thesis, homology modeling, molecular docking, molecular dynamics simulation, binding free energy calculation and biology experiments were integrated to investigate the structures, functions and modulations of several members of thioredoxin family, including human Erola, yeast Erolp, human QSOX as well as transcriptional repressor Sr-Rex which is sensitive to the concentration of NAD+and NADH. Besides, we also investigated the enantioselectivite mechanism of epoxide hydrase.In the first chapter, we simply introduced the background knowledge of oxidative stress and the relevant proteins, as well as computer aided drug design techniques which were used in this thesis.In the second chapter, the structural model of Erola was constructed by homology modeling. The RMSD of the model with the crystal structure which was published recently was1.09A. Detailed comparison with the structures of yeast Ero1p, Ero1α was predicted to be more active in alkaline environment but not as stable as Erolp, and the prediction results were validated by biological assays. We also investigated the position and critical features of Ero1α active pocket by active site prediction method. Based on these results, structure-based virtual screening towards Ero1p was implemented. Finally,12out of81compounds purchased from SPECS were confirmed to have micromolar inhibitions against Erolp, corresponding to a hit rate of about15%. And three of the12active compounds could selectively inhibit Ero1p.In the following chapter, we studied QSOX, another member of thioredoxin family, by homology modeling, protein-protein docking and binding free energy calculation. The function characteristics and interaction modes of QSOX were discussed and a novel electron transfer pathway was proposed. QSOX is a multidomain sulphydryl oxidase. The TRX1domain is homologous with a’domain of protein disulfide isomerase (PDI), and the Erv/ALR domain is homologous with the structures of Erv2p, another FAD-dependent sulfhydryl oxidase that can promote disulfide bond formation. Thus, QSOX contains the structural features and function characteristics of both Erv2p and PDI. The Erv/ALR domain of QSOX generates disulfide bonds using oxygen as its preferred terminal electron acceptors, and may shuttle electrons between substrate protein dithiols and the disulfide located around FAD.In the fourth chapter, we mainly investigated the interaction modes of Sr-Rex and its substrates in order to provide some clues for further biodynamics research. The redox-sensing repressor Rex binds NAD+or NADH as cofactors. Rex binds NAD+or NADH based on intracellular NADH/NAD+redox ratio, and then regulates the transcription of respiratory genes. By molecular dynamics, we investigated the interaction modes and the binding free energy of Sr-Rex with Rex operator site (ROP) and Sr-Rex with NADH/NAD+.Besides the oxidoreductases, we also investigated the enantioselectivite mechanisms of epoxide hydrolase (EH) from Bacillus megaterium in the fifth chapter. The results suggested that the hydrolysis of racemic epoxides catalyzed by EH was highly enantioselective. For methyl-substituted phenyl glycidyl ethers and nitro-substituted phenyl glycidyl ethers, the activity of EH was somewhat different. Here, we analyzed the docking poses in detail by measuring the distances and angles formed between the atoms of substrates and those of reactive residues to shed light on the enantioselective mechanisms. It was concluded that the distance of the oxygen atom of Asp used for nucleophilic attack and the catalytically active carbon atom of substrates could influence the activity of EH significantly, and the distance difference contributed dramatically to the selective strength.