1 Scientific RepoRts | 7:42494 | DOI: 10.1038/srep42494 www.nature.com/scientificreports Arabidopsis thaliana dehydroascorbate reductase 2: Conformational fexibility during catalysis Nandita Bodra 1,2,3,4,5,* , David Young 1,2,3,* , Leonardo Astolf Rosado 1,2,3 , Anna pallo 1,2,3 , Khadija Wahni 1,2,3 , Frank De proft 6 , Jingjing Huang 1,2,3,4,5 , Frank Van Breusegem 4,5 & Joris Messens 1,2,3 Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational diferences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic fndings, we investigated potential conformational fexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release. Oxidative stress has a signifcant impact on the cellular environment of organisms. Control of the reactive oxy- gen species (ROS) that cause such stress is essential for efective redox homeostasis. Generation of ROS can occur endogenously through leakage from respiratory complexes or photosystems, or can be induced by exter- nal stressors, such as UV radiation, drought, temperature extremes, or elevated salinity 1–5 . Once released, ROS infict cellular damage through oxidative inactivation of enzymes, metal oxidation, and mutagenesis 6,7 . Soluble small-molecule antioxidants, such as ascorbate (AsA) or glutathione (GSH), neutralize ROS either by direct reduction or by acting as cofactors for redox enzymes, such as peroxidases 8–10 . Cellular compartments maintain a reducing environment by constant recycling of oxidized antioxidants back to their reduced forms, a reaction catalyzed by glutathione reductase (GR) in the case of oxidized glutathione (GSSG) and dehydroascorbate reduc- tase (DHAR) for dehydroascorbate (DHA), the oxidized form of AsA 11 . Te intracellular concentration of GSH and AsA in plants are typically maintained within the range of 2–6 mM and 2–25 mM, respectively. GSH (5 mM) is able to directly reduce DHA through a non-enzymatic mechanism, albeit at a rate of 17 nmol min -1 12,13 , which is signifcantly lower than the reduction catalyzed by DHAR (20–370 μmol min -1 mg -1 ) 14 . AsA typically behaves as a single-electron donor and is converted to its semi-oxidized radical form, mono- dehydroascorbate (MDHA) upon ROS reduction. Two molecules of MDHA then disproportionate into AsA and DHA or, alternatively, MDHA can be enzymatically reduced to AsA by MDHA reductase 15 . Whereas GSH is relatively stable in its oxidized form, DHA undergoes irreversible hydrolysis to diketogluonate (DKG) 16 , and therefore, rapid reduction of DHA in cells is critical for efective AsA recycling. AsA is the major antioxidant of plants and, accordingly, the majority of the characterized DHAR enzymes are of plant origin. Plant DHAR 1 Center for Structural Biology, VIB, 1050 Brussels, Belgium. 2 Brussels Center for Redox Biology, 1050 Brussels, Belgium. 3 Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium. 4 Center for Plant Systems Biology, VIB, 9052 Gent, Belgium. 5 Department of Plant Biotechnology and Bioinformatics, Ghent University, 9052 Gent, Belgium. 6 Research Group of General Chemistry, Vrije Universiteit Brussel, 1050 Brussels, Belgium. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to J.M. (email: joris.messens@vib-vub.be) Received: 19 August 2016 Accepted: 11 January 2017 Published: 14 February 2017 opeN