Non-native ligands define the active site of Pennisetum glaucum (L.) R. Br dehydroascorbate reductase

• Published in: Biochemical and biophysical research communications 2016-05, Vol.473 (4), p.1152-1157
• Main Authors: Krishna Das, Bhaba, Kumar, Amit, Maindola, Priyank, Mahanty, Srikrishna, Jain, S.K., Reddy, Mallireddy K., Arockiasamy, Arulandu
• Format: Article
• Language: English
• Subjects: acetates; Active site; active sites; Ascorbate; ascorbic acid; Ascorbic Acid - chemistry; Binding Sites; Cenchrus americanus; crystal structure; Dehydroascorbate reductase; Enzyme Activation; Glutathione; glutathione dehydrogenase (ascorbate); glutathione transferase; Glutathione Transferase - chemistry; Glutathione Transferase - ultrastructure; glycerol; Ligands; Molecular Docking Simulation; Non-native ligands; Pennisetum - metabolism; Pennisetum glaucum; Plant Proteins - analysis; Plant Proteins - chemistry; Protein Binding; Protein Conformation; Substrate Specificity
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2016.04.031

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. •Crystal structure of plant dehydroascorbate reductase bound to non-native ligands.•Acetate and glycerol bound to G and H-sites mimic native ligands GSH and AsA/DHA.•Non-native ligands define the ‘hook points’ in Pennisetun glaucum DHAR active site.•Conformational flexibility of GSH bound to G-site is defined using a large data set.•Our data will aid in bootstrapping fragment based inhibitor/analog design for DHARS.