Fig. 4

Desmethylglycitein (DMG) inhibits AeNobo. A Chemical structures of DMG, also known as 4′,6,7-trihydroxyisoflavone. B Inhibition of the GSH conjugation activities of wild-type AeNobo (WT, blue dots and black solid curves) and the mutated AeNobo substituting Glu-113 with Ala (E113A, red dots) using 3,4-DNADCF in the presence of DMG. Each relative activity is defined as the ratio of activity compared between the respective proteins without DMG. All of the data points in duplicate assays are indicated. C Amino acid residues interacting with DMG. Carbon atoms of DMG are colored gray. Oxygen, nitrogen, and sulfur atoms are colored red, blue, and yellow, respectively. A water molecule interacting with each ligand is represented with a yellow sphere. Amino acid residues located within a 4.0-Å radius of the nearest atom of the flavonoids are shown. Additionally, amino acid residues that form hydrogen bonds within a 3.3-Å radius of the nearest atom of the flavonoids are also shown. Hydrogen bonds are illustrated by dashed yellow lines. The two views are related by a 180° rotation around the bold black line axis. Note that the hydrogen bond interaction between the hydroxyl residue of the B-ring and Arg-41 in chain D is indicated in this figure, while the direct interaction between DMG and Arg-41 is not observed in chains A, B, or C. C′ The hydrogen bonds between Glu-113 and the hydroxyl residues of C6 and C7 of the A-ring of DMG are highlighted. D, E In silico evaluation of the contribution of Glu-113 to the interaction between AeNobo and DMG. MD simulations of the AeNobo-WT or AeNobo-E113A complex with GSH and DMG in a SPC-water model were conducted at 300 K for 1000 ns. These simulations were performed in triplicate. D MD models at several representative time points of AeNobo-WT and AeNobo-E113A with DAI. The lower models are rotated 90° from the upper models. E RMSD of DMG heavy atoms in the MD simulations. Green: WT; blue: E113A mutation model. F Distance between the carboxylate’s C atom of Glu-113 of AeNobo-WT or Cβ of Ala-113 of AeNobo-E113A and the O6 or O7 atom of DMG at each frame. The nearest distances between O6 and O7 atoms are represented in this graph