Fig. 2: Functional characterization and predicted evolutionary trajectory of DAs, DSs, and OCs.

a The schematic diagram depicts the biosynthetic enzymes of chalcomoracin, moracin D, and 6 in Morus alba. OCs catalyze the oxidative cyclization reaction of moracin C (1) to form moracin D (2) (reaction A), while DSs catalyze the oxidative dehydrogenation reaction of moracin C (1) to give diene 3 (reaction B). The biosynthesis of chalcomoracin occurs through the Diels–Alder reaction between dienophile 4 and diene 3 (reaction C). b A phylogenetic tree provides insight into the evolutionary relationship between Diels-Alderases (DAs, orange), Diene Synthases (DSs, green), and FAD-dependent oxidocyclases (OCs, light purple) in Morus alba. Enzymes labeled with blue asterisks (i.e. ancDADS and MaDSs) can recognize morachalcone A (4) as substrate. The abbreviations “Ma” and “Mn” represent Morus alba and Morus notabilis, respectively. c Relative activities of these three types of enzymes. HPLC analysis was employed to quantify the relative enzymatic activities of OCs (reaction A), DSs (reaction B), and DAs (reaction C). These activities are represented by light purple, turquoise, and orange, respectively. The enzyme assay for detecting D-A activity was conducted with 5 μg of purified protein (0.1–2 mg/mL), 1 μL of diene 3 (100 μM), 1 μL of morachalcone A (4) (100 μM) and a 20 mM Tris-HCl solution at pH 8.0. The enzyme assay conditions for detecting oxidative activity included: 5 µg of purified protein, 1 µL of moracin C (1) (100 μM), and 20 mM Tris-HCl, pH 8.0. The mixture was then incubated at 50 °C for 7 min. The data are presented as mean values ± standard error (SE), with error bars indicating the standard deviations of three independent measurements. d Inferred evolutionary trajectories. Circles of different colors correspond to distinct enzyme types, while circles with two colors indicate bifunctional enzymes.