Fig. 3: The structure, binding pockets, and key mutant residues of DAs.

a The crystal structure of the MaDA1 (PDB ID: 7YAV) is a dimer. One chain is depicted as a cartoon, while the other appears as a gray surface. The cofactor FAD is shown in orange. b Superimposition of the structures of ancDA, ancDADS, and MaDA1. The active pockets of these enzymes are shown in different colors. The structures of ancDA and ancDADS were predicted by AlphaFold. c The binding pocket of ancDA. The transition state (endo-5) of the substrate appears in purple. The purple dashed line indicates the site of the chemical reaction, while the black dashed line represents the interactions between the substrates and the surrounding residues. The nonconserved residues are highlighted in red or blue font, while the conserved residues are shown in black. d Sequence logos illustrate the 21 different residues between DAs and OCs. e Diels–Alder activity of ancDA and its mutants, using diene 3 and morachalcone A (4) as substrates. The Diels–Alder reaction was conducted in 100 μl of 20 mM Tris-HCl buffer containing 5 μg of the mutants, with 1 μL of dienophiles 4 (100 μM) and 1 μL of dienes 3 (100 μM). The reaction took place at pH 8.0 and 70 °C for 7 min. Relative activity was measured using UPLC and calculated, considering the activity of wild-type and to be 100%. “ND” signifies not detected. The data are presented as mean values ± standard error (SE), with error bars indicating the standard deviations of three independent measurements.