Fig. 2

Glu-113 is essential for the binding of AeNobo to daidzein and luteolin. A, B The hydrogen bonds between Glu-113 and the hydroxyl residues of C7 of the A-ring of daidzein (DAI; A) and of C3′ and C4′ of the B-ring of luteolin (B) are highlighted from Fig. 1G and H, respectively. Carbon atoms of daidzein and luteolin are colored orange and light violet, respectively. Oxygen, nitrogen, and sulfur atoms are colored red, blue, and yellow, respectively. C, D 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 DAI (C) and luteolin (D). Each relative activity is defined as the ratio of activity compared between the respective proteins without the flavonoids. All the data points in duplicate assays are indicated. E, F In silico evaluation of the contribution of Glu-113 to the interaction between AeNobo and DAI. MD simulations of the AeNobo-WT or AeNobo-E113A complex with GSH and DAI in a SPC-water model were conducted at 300 K for 1000 ns. These simulations were performed in triplicate. E 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. F RMSD of DAI heavy atoms in the MD simulations. Green: WT; blue: E113A mutation model. G Distance between the carboxylate C atom of Glu-113 of AeNobo-WT or Cβ of Ala-113 of AeNobo-E113A and the O7 atom of DAI at each frame