Molecular Design And Thereotical Study Of Highly Efficient And Smart Antioxidant Seienoenzymes

Taking advantage of the allosteric characteristic of the calcium-binding protein (re-coverin), a Ca2+-responsive artificial selenoenzyme was constructed by computationaldesign and engineering of recoverin with a glutathione peroxidase (GPx)-like activecenter. Structural analysis of the switching conformation between “on” and “off” statesof the engineered catalytic site in combination with theoretical investigation of the mo-lecular recognition machanism of wild recoverin and its variants with the native sub-strate glutathione (GSH) of GPx will help to guide the future design and constructionof smart, efficient antioxidant selenoenzyme by site-directed mutagenesis. The mainresearch work includes the following two parts:1. The design of Ca2+-responsive GPx mimic by molecular docking.Reactive oxygen species (ROS) attack cell to cause different degrees of oxidativedamage tightly associated with cytosolic free calcium concentration. Inspired by thisphenomenon, a Ca2+-responsive allosteric protein, recoverin, was chosen as a scaffoldto construct the smart GPx mimic that has the ability to spontaneously respond to theconcentration change of ROS. First, molecular docking was employed to screen out thelow-energy binding mode between the allosteric region of recoverin and GSH. Thecatalytic site for selective modification with selenocysteine (Sec) was identified bycombining the recognition capacity for GSH and steric orientation of catalytic sele-nium moiety. Then, structural analysis of the conformational change of recoverinupon Ca2+binding were investigated in detail to elucidate the intrinsic correlation be-tween the “on/off” states of Sec and allosteric effect of recoverin. Moreover, some keyresidues and their contributions to substrate recognition such as hydrogen bond andelectrostatic interaction were further investigated, which will provide a theoreticalfoundation for the preparation of a Ca2+-responsive artificial selenoenzyme.2. Investigation of the substrate recognition mechanism of the smart GPx mimicby molecular dynamics and free energy calculation Theoretical studies of the dynamic structures and thermodynamic stabilities of en-zyme-substrate complexes and the contributions from these key residues during therecognition process are very important to explore the recognition mechanism and fur-ther improve the catalytic efficiency of the smart GPx mimic. Based on the abovedocking results, three mutations including Q50R, A64G and H68N were chosen to in-vestigate their influences on the recognition capacity of this GPx mimic with GSH.MD simulations combined with MM-PBSA free energy calculations was performed toanalyze the structural changes vs simulation time and the corresponding thermodynam-ic parameters (ΔG, ΔH, and ΔS) for eight sets of recoverin-GSH complexes: WT-re-coverin, single mutation models (recoverin-Q50R, recoverin-A64G and recoverin-H68N), double mutations models (recoverin-Q50R/A64G, recoverin-A64G/H68N andrecoverin-Q50R/H68N), and three mutations model (recoverin-Q50R/A64G/H68N). Afurther comparison of the structural complementary, surface charge distribution, andthe calculated binding free energy among the eight systems could evaluate the differ-ences of their binding affinity and stability. Furthermore, the binding free energies de-composition analysis and structural analysis indicated that the forces driving the rec-ognition of these binding events are all enthalpy-drived, which van der Waals interac-tion is the primary driving force to direct the recognition events and secondary interac-tions such as hydrogen bonding, hydrophobic interaction and electrostatic interactionhave a synergistic influence on these processes. In order to determine which residue isresponsible for the enzyme-substrate recognition, the free-energy decomposition ofeach residue in the active site, especially the key residues Arg50and Gly64, was con-ducted to assess the individual contribution to the binding free energy. These findingsdescribed here could provide better understanding of the efficiency and selectivity ofrecognition affected by mutations, which favors the rational design of substrate bind-ing specificity of the smart GPx mimic to enhance their catalytic activity.