Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis

Nandita Bodra,Frank De Proft,David Young,Khadija Wahni,Joris Messens,Frank Van Breusegem,Jingjing Huang,Anna Pallo,Leonardo Astolfi Rosado

doi:10.1038/srep42494

Nandita Bodra, Frank De Proft + Show 7 more

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https://doi.org/10.1038/srep42494

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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 differences 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 findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release.

Highlights

Oxidative stress has a significant impact on the cellular environment of organisms
Four independent structures of plant Dehydroascorbate reductase (DHAR) have been deposited in the Protein Data Bank: the crystallographic structures of Oryza sativa (OsDHAR1; PDB, 5D9T)[18], Pennisetum glaucum (PgDHAR1; PDB, 5EV0, 5IQY), the nuclear magnetic resonance solution structure of DHAR3A from Populus trichocarpa (PtDHAR3A; PDB, 2N5F)[19], and the recently deposited crystal structure of Arabidopsis thaliana DHAR1 (AtDHAR1; PDB, 5EL8)[20]
Crystal structures of P. trichocarpa GST Lambda (PtGSTL)[21] and Homo sapiens GST Omega (HsGSTO)[22] with DHAR activity have been determined with GSH bound at the catalytic cysteine

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Introduction

Oxidative stress has a significant impact on the cellular environment of organisms. Control of the reactive oxygen species (ROS) that cause such stress is essential for effective redox homeostasis. DHAR has been reported to have a bi-uni-uni-uni ping-pong enzymatic mechanism, with GSH and DHA interacting with the catalytic cysteine (Cys[20] in AtDHAR2) in separate, sequential binding events (Fig. 1)[27].

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