Insights into stereoselective ring formation in canonical strigolactone: Discovery of a dirigent domain-containing enzyme catalyzing orobanchol synthesis

Strigolactones (SLs) are plant apocarotenoids with diverse functions and structures. The widespread canonical SLs, with distinctive structural variations in their tricyclic lactone known as the ABC-ring, are classified into two types based on the C-ring configurations. The steric C-ring configuration arises during the BC-ring closure downstream of carlactonoic acid (CLA), a biosynthetic intermediate. Most plants stereoselectively produce either type of canonical SLs, e.g., tomato (Solanum lycopersicum) produces orobanchol with -oriented C-ring. The mechanisms governing SL structural diversification are partly understood, with limited insight into the functional implications. Moreover, the precise molecular mechanism for the stereoselective BC-ring closure reaction remains unknown. Herein, we identified an enzyme called the stereoselective BC-ring-forming factor (SRF) from the dirigent protein (DIR) family, especially the DIR-f subfamily, whose biochemical function was previously unidentified, making it a pivotal enzyme in stereoselective canonical SL biosynthesis with the -oriented C-ring. We begin by confirming the exact catalytic function of the tomato cytochrome P450 SlCYP722C, which we previously demonstrated to be involved in the orobanchol biosynthesis [Wakabayashi et al., Sci. Adv. 5, eaax9067 (2019)], to convert CLA to 18-oxocarlactonoic acid. Subsequently, we demonstrate that SRF catalyzes the stereoselective BC-ring closure reaction of 18-oxocarlactonoic acid to form orobanchol. Our approach integrates experimental and computational methods, including SRF structure prediction and molecular dynamics simulations, to propose a catalytic mechanism based on the conrotatory 4{pi}-electrocyclic reaction for stereoselective BC-ring formation in orobanchol. The present study provides insight into the molecular basis of how plants produce SLs with specific stereochemistry in a controlled manner.