Evolution of Chalcone Isomerase from a Non-Enzymatic Ancestor

Chalcone isomerase (CHI) is a key enzyme in plant flavonoid biosynthesis and catalyzes the conversion of an acyclic chalcone to the flavanone naringenin (k_cat/K_M > 10⁶ M^-1s^-1). For a long time, CHI’s evolutionary history remained a mystery, but recent evidence suggests that it evolved from a non-catalytic precursor. This ancestor relates to an older lineage of fatty-acid binding proteins (FAPs) as well as to another protein family of unknown function and with no CHI activity (CHILs for “CHI-like”).¹ Therefore, studying CHI’s evolution offers the unique opportunity to understand the emergence of catalysis in a non-enzymatic protein fold. Moreover, it can provide new insights into flavonoid metabolism, which is a target for the engineering of novel plants, e.g. with improved antioxidant properties.

We reconstructed the CHI family ancestor, as well as the older CHI/CHIL common ancestor. Using directed evolution, we retraced the transition from non-catalytic to catalytic ancestor and characterized increasingly active variants obtained along the trajectory by enzymology, protein crystallography, computational simulations, and complementation of CHI knockouts in Arabidopsis thaliana.

We found that the CHI/CHIL common ancestor has no detectable CHI activity. However, catalysis emerged within a single point mutation. Intriguingly, this and other mutations along the trajectory were minor exchanges between hydrophobic amino acids in the pre-active site, highlighting how subtle reshaping of an inert binding pocket can lay the foundation for an efficient enzyme. Computational simulations suggest that catalysis is triggered primarily by changes in the configuration of the active-site arginine. Future work is aimed at validating the evolving CHI activity in a living plant and at identifying the evolutionary origin and function of the CHIL family.

¹ Ngaki et al., Nature 2012, 485, 530-536