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Evolution of the chalcone-isomerase fold from fatty-acid binding to stereospecific catalysis

Abstract

Specialized metabolic enzymes biosynthesize chemicals of ecological importance, often sharing a pedigree with primary metabolic enzymes1. However, the lineage of the enzyme chalcone isomerase (CHI) remained unknown. In vascular plants, CHI-catalysed conversion of chalcones to chiral (S)-flavanones is a committed step in the production of plant flavonoids, compounds that contribute to attraction, defence2 and development3. CHI operates near the diffusion limit with stereospecific control4,5. Although 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. Arabidopsis thaliana contains five actively transcribed genes encoding CHI-fold proteins, three of which additionally encode amino-terminal chloroplast-transit sequences. These three CHI-fold proteins localize to plastids, the site of de novo fatty-acid biosynthesis in plant cells. Furthermore, their expression profiles correlate with those of core fatty-acid biosynthetic enzymes, with maximal expression occurring in seeds and coinciding with increased fatty-acid storage in the developing embryo. In vitro, these proteins are fatty-acid-binding proteins (FAPs). FAP knockout A. thaliana plants show elevated α-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’ enzyme6 from a non-enzymatic ancestor over a defined period of plant evolution.

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Figure 1: CHI fold and catalytic reaction.
Figure 2: Three-dimensional structure and ligand binding of FAPs.
Figure 3: Phenotypic characterization of Atfap1 null plants.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors are deposited in Protein Data Bank under accession numbers 4DOI (AtCHI), 4DOK (AtCHIL), 4DOL (AtFAP3) and 4DOO (AtFAP1).

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Acknowledgements

We thank A. Perera, B. Nikolau, H. Ilarsan and J. Peng for technical training (to M.N.N.), J. Peng for the fap1-1 homozygote mutant line, D. Nettleton and H. Wang for statistical analysis of initial seed fatty-acid data, and Eric Scheeff for assistance in Bayesian phylogenetic analysis. This research was supported in part by a Fulbright Fellowship (to M.N.N.). This material is based in part upon work supported by the National Science Foundation under award number MCB-0645794 (to J.P.N.), EEC-0813570 (to E.S.W.), MCB-0951170 (to E.S.W.), and by National Cancer Institute award number CA14195 (to G.M.) and the Plant Sciences Institute at Iowa State University (to E.S.W). J.P.N. is an investigator with the Howard Hughes Medical Institute. Portions of this research were conducted at the Advanced Light Source, a national user facility operated by Lawrence Berkeley National Laboratory, on behalf of the US Department of Energy, Office of Basic Energy Sciences. The Berkeley Center for Structural Biology is supported in part by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences. We thank the staff at the Advanced Light Source for assistance with X-ray data collection. Any opinions, findings and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

Author information

Authors and Affiliations

Authors

Contributions

M.N.N. experimentally characterized the FAP genes in planta. M.E.B., R.N.P. and E.L. expressed, purified and crystallized proteins. L.L. designed genetics experiments and constructs. G.V.L. and F.P. performed fatty-acid binding analyses and solved the X-ray crystal structures. R.N.P. performed thermal-shift assays of fatty-acid binding. G.M. and E.L. performed phylogenetic and sequence analyses. J.P.N. designed the biochemical experiments; E.S.W. designed the bioinformatics and functional genomics experiments. The manuscript was written by R.N.P., G.V.L., M.N.N., J.P.N., G.M. and E.S.W.

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Correspondence to Eve Syrkin Wurtele or Joseph P. Noel.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-23 and Supplementary Tables 1-9. (PDF 6495 kb)

Supplementary Data 1

This file contains CHI-fold Family Multisequence Alignment data. (TXT 107 kb)

Supplementary Data 2

This file contains CHI-fold Family Sequences and Accession Numbers. (XLS 83 kb)

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Ngaki, M., Louie, G., Philippe, R. et al. Evolution of the chalcone-isomerase fold from fatty-acid binding to stereospecific catalysis. Nature 485, 530–533 (2012). https://doi.org/10.1038/nature11009

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