Elucidating the Biosynthetic Pathway and Mechanisms of Retrochalcones

Chalcone is a privileged natural product skeleton for drug discovery, and retrochalcone represents a group of nonclassical chalcones with a distinctive oxygen substitution pattern. Echinatin, a hepatoprotective agent, is a retrochalcone derived from Glycyrrhiza inflata. Despite their initial discovery half a century ago, the biosynthetic mechanisms of retrochalcones have remained elusive. In this work, we identified a ketoreductase, GinKR1, which selectively catalyzes the reduction of the 1″-carbonyl group of the dibenzoylmethane precursor 2'-O-methyllicodione, followed by spontaneous dehydration to form the retrochalcone skeleton. Our findings reveal that the A and B rings of retrochalcones are derived from the shikimate and polyketide pathways, respectively, which are reversed to normal chalcones. In addition, ¹⁸O isotope labeling verifies that the carbonyl oxygen of retrochalcones is derived from the hydroxyl group introduced by a flavanone 2-hydroxylase. The complete biosynthetic pathway of echinatin was elucidated by identifying six enzymes from G. inflata. Moreover, we determined the crystal structure of GinKR1 and identified a critical α10 helix responsible for its regioselectivity. With this α10 helix as a marker, we further discovered homologous genes of GinKR1 from 185 plant species. This study elucidates the biosynthetic pathway and underlying mechanisms of retrochalcones.