Structural understanding of the recycling of oxidized ascorbate by dehydroascorbate reductase (OsDHAR) from Oryza sativa L. japonica

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.

Proteomic Responses to Drought Vary Widely Among Eight Diverse Genotypes of Rice (Oryza sativa)

“…Naturally, the eight proteins induced in response to drought in all genotypes (DHAR1, KAT, LTP1, HSP 18.6, GRXC6, RNS3, ADF3, and TIM18) are prime candidates for the selection of drought-tolerance markers. Most of these proteins have been reported to play important roles in response to plant hormones [14][15][16][17] and are involved in detoxification of reactive oxygen species (ROS) [15,18,19]. Protein transport to mitochondria through the membrane is facilitated by TIM complexes, potentially delivering antioxidant enzymes to mitochondria and enabling ROS detoxification during water deficits.…”

Expression of Heterologous OsDHAR Gene Improves Glutathione (GSH)-Dependent Antioxidant System and Maintenance of Cellular Redox Status in Synechococcus elongatus PCC 7942

“…OsDHAR also contains interacting residues of the GSH binding site. This indicates that OsDHAR can supply a redox active site during ROS-induced oxidative stress (Pulido et al, 2010;Do et al, 2016;Kim et al, 2017). The similarity of the aforementioned conserved DHAR sequences indicated that the OsDHAR redox active site possesses strictly conserved residues, including a GSH binding site and similar functional conserved residues from cyanobacteria GSTs.…”

“…Thus, DHAR as isozymes are critical to cellular detoxification of exogenous and endogenous harmful compounds. In aerobic cells, high DHAR activity enhances growth and development through increased chlorophyll fluorescence under oxidative stress, whereas it decreases chlorophyll in aging cells under the same conditions (Wang et al, 2010;Kim et al, 2013;Do et al, 2016). In cyanobacteria, H 2 O 2 -induced oxidative stress and the resulting excess ROS lead to cell death in several types of cyanobacteria (Drabkova et al, 2007;Qian et al, 2010;Banerjee et al, 2015).…”

“…In previous studies, we initially predicted that the GSH-bound Oryza sativa L. japonica DHAR (OsDHAR) structure might contain a GSH binding site (Do et al, 2016). A subsequent study reported the pronounced similarity of GSH binding sites in the conserved residues of glutathione S-transferases (GSTs), such as thioredoxin-like sites found in different species (Abdul Kayum et al, 2018).…”

“…In DHAR activity, the first cysteine residue in the GSH binding site reacts with the sulfur atom in the GSH mixed disulfide bridges and promotes thiol transfer (Dixon et al, 2002;Dixon and Edwards, 2010). Mutation of the first cysteine residue induces significant losses of catalytic efficiency and GSH binding affinity at GSH binding site residues during abiotic stresses (Tang and Yang, 2013;Do et al, 2016).…”