Fig. 1

Identification and characterization of daidzein and luteolin as flavonoids that inhibit the AeNobo enzymatic activity and interact with H-sites of AeNobo. A Schematic of the library screen to identify chemical compounds that inhibit AeNobo in vitro with IC₅₀ values of less than 10 μM. One of the identified compounds was 2′-hydroxyflavanone. B Schematic of a screen to identify flavonoid compounds that inhibit AeNobo in vitro with IC₅₀ values of less than 10 μM. The IC₅₀ values of the nine tested compounds, including daidzein and luteolin, were less than 10 μM. C, D Chemical structures of daidzein (C) and luteolin (D). E, F Inhibition of the GSH conjugation activities of AeNobo with an artificial fluorescent substrate, 3,4-DNADCF, in the presence of daidzein (E) and luteolin (F). 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. G, H Amino acid residues interacting with daidzein (G) and luteolin (H). 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. 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