Enzymatic characterization and crystal structure analysis of Chlamydomonas reinhardtii dehydroascorbate reductase and their implications for oxidative stress.

摘要: Dehydroascorbate reductase (DHAR) is a key enzyme for glutathione (GSH)-dependent reduction of dehydroascorbate (DHA) to recycled ascorbate (AsA) in plants, and plays major role against the toxicity reactive oxygen species (ROS). Previously, we proposed that increase AsA regeneration via enhanced DHAR activity modulates ascorbate-glutathione cycle photooxidative stress Chlamydomonas reinhardtii. In present work, use site-directed mutagenesis crystal structure analysis elucidate molecular basis how C. reinhardtii (CrDHAR1) involved detoxification mechanisms. Mutagenesis data show D21A, D21N C22A mutations result severe loss enzyme's function, suggesting crucial roles Asp-21 Cys-22 substrate binding catalysis. The mutant K11A also exhibits reduced redox (∼50%). apo CrDHAR1 further provides insights into mechanism centering on strictly conserved Cys-22, which suggested initiate reactions DHA GSH. Furthermore, in vitro oxidation recombinant presence 1 mM H2O2 has minor effects Km substrates but significantly reduces kcat. its mRNA abundance cells are increased by treatment with 0.2-1 mM decreased when is ≥ 1.5 mM. latter decrease accompanied oxidative damage lower concentrations. These biochemical physiological provide new catalytic CrDHAR1, protects from stress-induced toxicity.