Evolution of the Chalcone Isomerase Fold from Fatty Acid-Binding to Stereospecific Enzyme

Specialized metabolic enzymes biosynthesize chemicals of ecological importance, often sharing a pedigree with primary metabolic enzymes(1). However, the lineage of the enzyme chalcone isomerase (CHI) remained a quandary. In vascular plants, CHI-catalyzed conversion of chalcones to chiral (S)-flavanones is a committed step in the production of plant flavonoids, compounds that contribute to attraction, defense(2), and development(3). CHI operates near the diffusion limit with stereospecific control(4,5). While associated primarily with plants, the CHI-fold occurs in several other eukaryotic lineages and in some bacteria. Here we report crystal structures, ligand-binding properties, and in vivo functional characterization of a non-catalytic CHI-fold family from plants. A. thaliana contains five actively transcribed CHI-fold genes, three of which additionally encode amino-terminal chloroplast-transit sequences (cTP). These three CHI-fold proteins localize to plastids, the site of de novo fatty acid (FA) biosynthesis in plant cells. Furthermore, their expression profiles correlate with those of core FA biosynthetic enzymes, with maximal expression occurring in seeds and coinciding with increased FA storage in the developing embryo. In vitro, these proteins are Fatty Acid-binding Proteins (FAP). FAP knockout A. thaliana plants exhibit elevated alpha-linolenic acid levels and marked reproductive defects, including aberrant seed formation. Notably, the FAP discovery defines the adaptive evolution of a stereospecific and catalytically ‘perfected’ enzyme(6) from a non-enzymatic ancestor over a defined period of plant evolution.