Snapshots From the Catalytic Landscape of Chalcone Isomerase.

Chalcone isomerase (CHI) catalyzes the cyclization of three-ring scaffolds of flavonoids, a class of plant-based natural products important for nutrition and disease prevention. A persistent question has been whether the enzyme uses protein dynamics to facilitate conformational rearrangements of substrates within the active site. To help resolve this question, CHI from Medicago truncatula was crystallized with phloretin, a flexible substrate analogue that cannot undergo cyclization, and solved to 2.0-Å resolution. The crystal structure possesses eight protein molecules per asymmetric unit, revealing different active site conformations that accommodate different bound conformers of phloretin. Together, the structural snapshots depict a series of coordinated, dynamic chemical interactions that lower barriers to substrate rearrangements approaching bond formation. Differential scanning fluorimetry combined with mutational analysis and enzyme kinetics further confirms that phloretin binds to the enzyme active site, and that it acts as a competitive inhibitor of CHI. Together, these findings answer outstanding questions about the flexibility and dynamics of CHI catalysis, information that may be useful for future biosynthetic design and enzyme engineering goals. Overall, this work supports a catalytic model in which the CHI enzyme operates as a dynamic ensemble of structures necessary to facilitate catalytic substrate rearrangements.