title: Non-native ligands define the active site of Pennisetum glaucum (L.) R. Br dehydroascorbate reductase.
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 γ-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.
authors.affiliation: Membrane Protein Biology Group, ICGEB, Aruna Asaf Ali Marg, New Delhi, Delhi 110067, India; Department of Biotechnology, Jamia Hamdard University, New Delhi, Delhi, 110062, India.
authors.forename: Bhaba
authors.initials: B
authors.lastname: Krishna Das
authors.affiliation: Membrane Protein Biology Group, ICGEB, Aruna Asaf Ali Marg, New Delhi, Delhi 110067, India.
authors.forename: Amit
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Membrane Protein Biology Group, ICGEB, Aruna Asaf Ali Marg, New Delhi, Delhi 110067, India.
authors.forename: Priyank
authors.initials: P
authors.lastname: Maindola
authors.affiliation: Crop Improvement Group, ICGEB, Aruna Asaf Ali Marg, New Delhi, Delhi, 110067, India.
authors.forename: Srikrishna
authors.initials: S
authors.lastname: Mahanty
authors.affiliation: Department of Biotechnology, Jamia Hamdard University, New Delhi, Delhi, 110062, India; Department of Medical Biochemistry, HIMSR, Jamia Hamdard University, New Delhi, Delhi, 110062, India.
authors.forename: S K
authors.initials: SK
authors.lastname: Jain
authors.affiliation: Crop Improvement Group, ICGEB, Aruna Asaf Ali Marg, New Delhi, Delhi, 110067, India.
authors.forename: Mallireddy K
authors.initials: MK
authors.lastname: Reddy
authors.affiliation: Membrane Protein Biology Group, ICGEB, Aruna Asaf Ali Marg, New Delhi, Delhi 110067, India. Electronic address: sam@icgeb.res.in.
authors.forename: Arulandu
authors.initials: A
authors.lastname: Arockiasamy
mesh_terms: D001205:Ascorbic Acid / Q000737:chemistry*; D001665:Binding Sites; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000737:chemistry* / Q000648:ultrastructure*; D008024:Ligands; D062105:Molecular Docking Simulation; D031782:Pennisetum / Q000378:metabolism*; D010940:Plant Proteins / Q000032:analysis / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D008024:Ligands; D010940:Plant Proteins; D005982:Glutathione Transferase; D001205:Ascorbic Acid
keywords: Active site; Ascorbate; Dehydroascorbate reductase; Glutathione; Non-native ligands; Pennisetum glaucum