Fig. 5: Mutations analysis of GbCYP90J6 revealed critical amino acid residues within the substrate pocket that determine biflavonoid dimerization specificity.

a Results of alanine scanning within a 5 Å range of the 5 ligands of GbCYP90J6. The x-axis represented the mutagenesis sites of GbCYP90J6, and the y-axis showed the relative enzyme activity compared to the wild-type (WT). Each value was presented as mean ± S.D. (n = 3). Experiments were conducted in vivo using E. coli JM109(DE3). “nd” indicated no production of 1. All mutagenesis within a 5 Å range of the 5 ligands showed significant variation compared to the WT. Source data are provided as a Source Data file. b GbCYP90J6-apigenin (5D)-apigenin (5U) model generated by molecular docking and MD simulation, showing the spatial relationship between key residues identified by mutagenesis and the substrate molecules. Two ligands maintained a head-to-tail, anti-parallel configuration, with distance from C4’-OH of ligand 1 (5D) and C7”-OH of ligand 2 (5U) to the heme center of 2.2 Å and 4.9 Å, separately. The distance between C3’ of 5D and C8’ of 5U was 3.5 Å. Key sites from alanine scanning mutagenesis were highlighted in different colors. c Protein sequence alignment of gymnosperm CYP90J, ordered by the sequence of GbCYP90J6, revealing conservation patterns of functionally important residues across active and inactive enzymes.