Abstract
Dehydroascorbate reductase (DHAR) is a key enzyme involved in the recycling of ascorbate, which catalyses the glutathione (GSH)-dependent reduction of oxidized ascorbate (dehydroascorbate, DHA). As a result, DHAR regenerates a pool of reduced ascorbate and detoxifies reactive oxygen species (ROS). In previous experiments involving transgenic rice, we observed that overexpression of DHAR enhanced grain yield and biomass. Since the structure of DHAR is not available, the enzymatic mechanism is not well-understood and remains poorly characterized. To elucidate the molecular basis of DHAR catalysis, we determined the crystal structures of DHAR from Oryza sativa L. japonica (OsDHAR) in the native, ascorbate-bound, and GSH-bound forms and refined their resolutions to 1.9, 1.7, and 1.7 Å, respectively. These complex structures provide the first information regarding the location of the ascorbate and GSH binding sites and their interacting residues. The location of the ascorbate-binding site overlaps with the GSH-binding site, suggesting a ping-pong kinetic mechanism for electron transfer at the common Cys20 active site. Our structural information and mutagenesis data provide useful insights into the reaction mechanism of OsDHAR against ROS-induced oxidative stress in rice.
Highlights
Dehydroascorbate reductase (DHAR) is a key enzyme involved in the recycling of ascorbate, which catalyses the glutathione (GSH)-dependent reduction of oxidized ascorbate
The closest structural relative was found to be human CLIC1 (PDB code 1K0O), in which 156 Cα could be aligned with a root-mean-square deviation (RMSD) value of 0.93 Å, followed by GSTO1 from Homo sapiens (PDB code 3LFL) (122 aligned Cα with an RMSD of 2.07 Å) and the putative stringent starvation protein A from Burkholderia cenocepacia (PDB code 4QQ7) (122 aligned Cα with an RMSD of 2.14 Å)[12,15]
The dimer structure of CLIC1 has a large hydrophobic surface, which can be used for membrane incorporation and chloride ion channel formation[15,16,17]
Summary
The crystallization and preliminary X-ray diffraction data analysis of the apo-OsDHAR were performed as described previously[30]. We attempted to collect data for the structure of the complex with DHA (3 mM) by the soaking method using the apo-OsDHAR crystals. Lysozyme (LYS; 0.25 mg/mL), α -crystallin (CRY; 0.25 mg/mL; Sigma-Aldrich, Saint Louis, MO, USA), or OsDHAR (0.25 mg/mL) was added to catalase (CAT; 0.1 mg/mL) in a reaction mixture containing 10 mM phosphate buffer (pH 7.0), which was heated at 60 °C for 22 min. To examine the effects of mutations on chaperone-like activity, OsDHAR WT, K8A, C20A, or K47A (0.2 mg/mL) was added to CAT (0.1 mg/mL) in the same reaction mixture and heated at 60 °C for 22 min. Relative data are presented relative to the WT, which was defined as 100%
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