Non-native ligands define the active site of Pennisetum glaucum (L.) R. Br dehydroascorbate reductase Bhaba Krishna Das a, b , Amit Kumar a , Priyank Maindola a , Srikrishna Mahanty c , S.K. Jain b, d , Mallireddy K. Reddy c , Arulandu Arockiasamy a, * a Membrane Protein Biology Group, ICGEB, Aruna Asaf Ali Marg, New Delhi, Delhi 110067, India b Department of Biotechnology, Jamia Hamdard University, New Delhi, Delhi,110062, India c Crop Improvement Group, ICGEB, Aruna Asaf Ali Marg, New Delhi, Delhi, 110067, India d Department of Medical Biochemistry, HIMSR, Jamia Hamdard University, New Delhi, Delhi,110062, India article info Article history: Received 23 March 2016 Accepted 7 April 2016 Available online xxx Keywords: Ascorbate Dehydroascorbate reductase Pennisetum glaucum Active site Non-native ligands Glutathione abstract Dehydroascorbate reductase (DHAR), a member of the glutathione-S-transferase (GST) family, reduces dehydroascorbate (DHA) to ascorbate (AsA; Vitamin-C) in a glutathione (GSH)-dependent manner and in doing so, replenishes the critical AsA pool of the cell. To understand the enzyme mechanism in detail, we determined the crystal structure of a plant DHAR from Pennisetum glaucum (PgDHAR) using Iodide- Single Anomalous Dispersion (SAD) and Molecular replacement methods, in two different space groups. Here, we show PgDHAR in complex with two non-native ligands, viz. an acetate bound at the G- site, which resembles the g-carboxyl moiety of GSH, and a glycerol at the H-site, which shares the backbone of AsA. We also show that, in the absence of bound native substrates, these non-native ligands help define the critical ‘hook points’ in the DHAR enzyme active site. Further, our data suggest that these non-native ligands can act as the logical bootstrapping points for iterative design of inhibitors/analogs for DHARs. © 2016 Elsevier Inc. All rights reserved. 1. Introduction Aerobic organisms have evolved intricate strategies to combat oxidative stress in cells and thus negate oxidation-induced cellular damage [1,2]. Antioxidants, such as ascorbic acid (AsA), tocopherols and glutathione (GSH), and enzymes like catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reduc- tase (GR), monodehydroascorbate reductase (MDHAR) and dehy- droascorbate reductase (DHAR) play key roles in protecting cells from an otherwise potentially lethal oxidative stress [3e6]. In particular, the ascorbate-glutathione pathway enzyme DHAR (EC: 1.8.5.1) is essential for recycling ascorbate from its oxidized form DHA [7e9]. DHARs belong to the GST-omega (GST-U) family and have a conserved CXXC/S motif that constitutes the active site [10,11]. The N-terminal cysteine of this motif is critical for in vitro reduction of DHA [12,13]. Plant DHARs are encoded by a multi-gene family with at least two known isoforms, viz. cytosolic and chlo- roplastic and are also reported to be present in other cellular compartments [14]. Overexpression of plant DHAR results in increased AsA levels, and thus improve protection from oxidative damage [15e18]. Increased DHAR activity is also reported in response to various stress conditions including drought [19], tem- perature [20], osmosis [21] and heavy metals [22]. Thus, DHAR gene was extensively used to generate transgenic plants for enhanced stress tolerance and added nutritive values. Lately an expanding repertoire of human proteins displaying DHAR like activity have catapulted it significance in human health as well [23,24]. However, the expanding list of biochemically characterized plant DHARs [9,14,25e27], along with the recent availability of crystal structure of enzymatically inactive Cys-20 mutant of Oryza sativa DHAR (OsDHAR), in complex with GSH and AsA bound to H- site [28] (PDB: 5D9T, 3VLN), still do not help understand the DHAR active site and the enzyme mechanism, consistent with the known biochemistry. * Corresponding author. Membrane Protein Biology, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India. E-mail addresses: sam@icgeb.res.in, asamy001@gmail.com (A. Arockiasamy). Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc http://dx.doi.org/10.1016/j.bbrc.2016.04.031 0006-291X/© 2016 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications xxx (2016) 1e6 Please cite this article in press as: B. Krishna Das, et al., Non-native ligands define the active site of Pennisetum glaucum (L.) R. Br dehydroascorbate reductase, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.04.031