Structural insights into pterocarpan reductases unveil a universal ring-opening mechanism in plant biosynthesis of 4-(furan-2-yl) phenol derivatives

Pterocarpans and isoflavans are important phytoalexins, and demonstrate significant benefits to human health. Pterocarpan reductases (PTRs) catalyze the conversion of pterocarpans to isoflavans, while the catalytic mechanism remains unknown. Herein, we report six PTRs (GuPTR1–6) from Glycyrrhiza uralensis , together with the first PTR crystal structure (GuPTR1/(‒)-medicarpin/NADP + , 1.8 Å). Structural analysis and mutagenesis reveal that a lysine-mediated deprotonation of the 7-OH group triggers C‒O bond cleavage of pterocarpans in the furan ring-opening reactions. This mechanism also applies to similar ring-opening enzymatic reactions. Through ancestral sequence reconstruction, we obtained a multifunctional reductase N0, which could accept different types of 4-(furan-2-yl) phenol derivatives as substrates. This study not only unveils the catalytic mechanisms of PTRs, but also provides a powerful enzymatic tool for the synthesis of bioactive isoflavans.