Group Of An Oxidoreductase In Saccharomyces Cerevisiae Oxidative Stress Response In The Structure And Function Study

All aerobic organisms are exposed to reactive oxygen species(ROS),such as H₂O₂,superoxide anion(·O2^-)and hydroxyl radical(·OH),which are produced during normal aerobic metabolism or after exposure to radical-generating compounds. Accumulation of ROS will cause oxidative damage to various biological molecules, including proteins,nucleic acids,cell membranes and lipids,leading to various diseases such as cancer,aging and neurodegenerative disorders.Cells possess both enzymatic and non-enzymatic defense systems to protect their cellular constituents and maintain the intracellular redox homeostasis.The enzymatic system is mainly composed of the glutaredoxin system and thioredoxin system.To investigate the protein-protein interaction network during oxidative stress,crystallographic studies and biochemical analyses were applied to some key protein targets in both systems.We determined the crystal structure of glutathione reductase Glr1 in oxidized state.Although the overall structure and the active site are highly conserved,obvious variety was found at the interface of Glr1 monomers when superimposed against the homolog from E.coli or human.The non-conserved C239 is exposed to the solvent and accessible to GSH or GSSG enriched in a microenvironment around the Glr1 molecules,leading to its partial and transient glutathionylation,as primarily identified from the 2Fo-Fc electron density map and further confirmed by biochemical assays. Meanwhile N278 at the vicinity of NADP-binding pocket was artificially glycosylated when heterogeneously overexpressed in P.pastoris.The highly motile oligosaccharide chain linked to N278 of the recombinant Glr1 interferes with the entry of NADPH, which results in a dramatic increase of Km for NAPDH and a significant decrease of turnover number,when compared with the native protein.We also determined crystal structures of Grx1 in oxidized and glutathionylated forms,respectively.Structural analyses demonstrate that subsequent to the breakage of disulfide between Cys27 and Cys30 from oxidized to glutathionylated state,the conformation at the region from residue Thr25 to Cys30 is dramatically rearranged. Residues essential for GSH binding were figured out according to the glutathionylated Grx1 structure.Despite the high degree of homology between Grx1 and Grx2(64% identity),evidence has shown that they play distinct roles in normal growth and stress conditions.Furthermore we solved crystal structures of Grx2 in oxidized and reduced states,elucidating the redox-induced conformational rearrangements in the region from Thr59 to Lys65.Structural comparisons between reduced Grx2 and glutathionylated Grx1 indicated that the residue change from Asp89 in Grx1 to Ser123 in Grx2 caused their difference in GSH binding affinity,which was further confirmed by isothermal titration calorimetry.We determined the crystal structure of alkyl hydroperoxide reductase Ahpl in oxidized state.Intermolecular disulfide was found between Cys31 and Cys62'.Ahpl exists as a homodimer in solution,with an interface perpendicular to the centralβ-sheet.Mutants of C31S,C62S and C120S were applied to SDS-PAGE to check the formation of intermolecular disulfide combined with identification in catalytic activities.Ahpl was found covalently modified by ubiquitin-related modifier Urml.It was proposed that the conjugation of Urmlp to Ahplp could regulate the function of Ahplp in antioxidant stress response in Saccharomyces cerevisiae.We determined its crystal structure and found the orientation of C-terminus is different with that in solution structure.The C-terminal tail twists and covers the region between strandsβ4 andβ5 in the crystal structure,mainly contributed to the dimeric interactions.The calculated molecular weight of Urml in solution is twice of the theoretical MW of the monomer.Furthermore renaturation between Urml(11.0 kDa)and Urml-6xHis-tag (13.1 kDa)was applied to check the existence of heterodimeric Urml in solution.