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Unformatted text preview: 1 Evolution of the Chalcone Isomerase Fold from Fatty Acid-Binding to Stereospecific Enzyme Micheline N. Ngaki 1 *, Gordon V. Louie 2 *, Gerard Manning 3 , Florence Pojer 2 , Marianne E. Bowman 2 , Elise Larsen 2 , Eve Syrkin Wurtele 1 & Joseph P. Noel 2 1. Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, Iowa 50011, USA 2. Howard Hughes Medical Institute, Jack H. Skirball Center for Chemical Biology and Proteomics, The Salk Institute for Biological Studies, La Jolla, California 92037, USA 3. Razavi Newman Center for Bioinformatics, The Salk Institute for Biological Studies, La Jolla, California 92037, USA *These authors contributed equally to this manuscript. Correspondence to: Eve Syrkin Wurtele 1 Email: firstname.lastname@example.org Correspondence to: Joseph P. Noel 2 Email: email@example.com Specialized metabolic enzymes synthesize diverse natural chemicals of ecological importance and often share a pedigree with primary metabolic enzymes 1 . However, the lineage of the specialized metabolic enzyme chalcone isomerase (CHI) remains a quandary. In vascular plants, CHI converts chalcones to chiral ( S )-flavanones, a committed step in plant flavonoid assembly that contributes to attraction and defense 2 and mediates development 3 . CHI operates near the diffusion limit with exquisite stereospecific control 4,5 . While associated with plants, the CHI-fold occurs in several eukaryotic lineages and in some bacteria. Here we report the crystal structures, ligand-binding properties and in vivo function of a non-catalytic CHI-fold family from plants. A. thaliana contains five actively transcribed and translated CHI-fold genes, three of which also encode amino-terminal chloroplast-transit peptides (cTP). These three localize to the plastidic site of de novo fatty acid (FA) biosynthesis in plant cells, and their expression correlates with core FA biosynthetic enzymes. Maximal expression occurs in seeds and coincides with peak generation of FA stores in the developing embryo. In vitro , these three CHI-fold proteins are FA-binding (FAB) proteins. FAB knockout A. thaliana plants contain elevated alpha- linolenic acid and exhibit reproductive defects, including aberrant seed formation. These results suggest FABs offer avenues for altering FA composition of foods and FA-derived biorenewables, while their discovery is a model for the adaptive evolution of a stereospecific and catalytically perfected enzyme 6 from a non-enzymatic ancestor over a defined period of plant evolution. CHI (EC 188.8.131.52) catalyzes the intramolecular and stereospecific cyclization of chalcones to chiral flavanones via a Michael addition reaction 7 . The evolutionary origin of CHI has been a mystery, due to the apparent absence of a related protein from primary metabolism 8 . 2 Homologues of CHI occur in fungi and bacteria 9 , but these lack key catalytic residues and the chalcone-binding site of bona fide CHI (Fig. 1a-d). Our phylogenetic analysis shows that CHI is chalcone-binding site of bona fide CHI (Fig....
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