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


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).


  1. Hartmann, T. From waste products to ecochemicals: fifty years research of plant secondary metabolism. Phytochemistry 68, 2831–2846 (2007)

    Article  CAS  Google Scholar 

  2. Ferrer, J.-L., Austin, M. B., Stewart, C., Jr & Noel, J. P. Structure and function of enzymes involved in the biosynthesis of phenylpropanoids. Plant Physiol. Biochem. 46, 356–370 (2008)

    Article  CAS  Google Scholar 

  3. Peer, W. A. & Murphy, A. S. Flavonoids and auxin transport: modulators or regulators? Trends Plant Sci. 21, 556–563 (2007)

    Article  Google Scholar 

  4. Bednar, R. A. & Hadcock, J. R. Purification and characterization of chalcone isomerase from soybeans. J. Biol. Chem. 263, 9582–9588 (1988)

    CAS  PubMed  Google Scholar 

  5. Jez, J. M. & Noel, J. P. Reaction mechanism of chalcone isomerase: pH dependence, diffusion control, and product binding differences. J. Biol. Chem. 277, 1361–1369 (2002)

    Article  CAS  Google Scholar 

  6. Albery, W. J. & Knowles, J. R. Evolution of enzyme function and the development of catalytic efficiency. Biochemistry 15, 5631–5640 (1976)

    Article  CAS  Google Scholar 

  7. Jez, J. M., Bowman, M. E. & Noel, J. P. Role of hydrogen bonds in the reaction mechanism of chalcone isomerase. Biochemistry 41, 5168–5176 (2002)

    Article  CAS  Google Scholar 

  8. Jez, J. M., Bowman, M. E., Dixon, R. A. & Noel, J. P. Structure and mechanism of the evolutionarily unique plant enzyme chalcone isomerase. Nature Struct. Biol. 7, 786–791 (2000)

    Article  CAS  Google Scholar 

  9. Gensheimer, M. & Mushegian, A. Chalcone isomerase family and fold: no longer unique to plants. Protein Sci. 13, 540–544 (2004)

    Article  CAS  Google Scholar 

  10. Shirley, B. W., Hanley, S. & Goodman, H. M. Effects of ionizing radiation on a plant genome: analysis of two Arabidopsis transparent testa mutations. Plant Cell 4, 333–347 (1992)

    Article  CAS  Google Scholar 

  11. Oursel, D. et al. Identification and relative quantification of fatty acids in Escherichia coli membranes by gas chromatography/mass spectrometry. Rapid Commun. Mass Spectrom. 21, 3229–3233 (2007)

    Article  CAS  Google Scholar 

  12. Niesen, F. H., Berglund, H. & Vedadi, M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nature Protocols 9, 2212–2221 (2007)

    Article  Google Scholar 

  13. Mentzen, W. I. & Wurtele, E. S. Regulon organization of Arabidopsis . BMC Plant Biol. 8, 99 (2008)

    Article  Google Scholar 

  14. Mentzen, W. I., Peng, J., Ransom, N., Nikolau, B. J. & Wurtele, E. S. Articulation of three core metabolic processes in Arabidopsis: fatty acid biosynthesis, leucine catabolism and starch metabolism. BMC Plant Biol. 8, 76–90 (2008)

    Article  Google Scholar 

  15. Ruuska, S. A., Girke, T., Benning, C. & Ohlrogge, J. B. Contrapuntal networks of gene expression during Arabidopsis seed filling. Plant Cell 14, 1191–1206 (2002)

    Article  CAS  Google Scholar 

  16. Falcone, D. L., Ogas, J. P. & Somerville, C. R. Regulation of membrane fatty acid composition by temperature in mutants of Arabidopsis with alterations in membrane lipid composition. BMC Plant Biol. 4, 17–31 (2004)

    Article  Google Scholar 

  17. Murakami, Y., Tsuyama, M., Kobayashi, Y., Kodama, H. & Iba, K. Trienoic fatty acids and plant tolerance of high temperature. Science 287, 476–479 (2000)

    Article  CAS  ADS  Google Scholar 

  18. Austin, M. B. & Noel, J. P. The chalcone synthase superfamily of type III polyketide synthases. Nat. Prod. Rep. 20, 79–110 (2003)

    Article  CAS  Google Scholar 

  19. Hilson, P. et al. Versatile gene-specific tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications. Genet. Res. 14, 2176–2189 (2004)

    Article  CAS  Google Scholar 

  20. Bonaventure, G., Salas, J. J., Pollard, M. R. & Ohlrogge, J. B. Disruption of the FATB gene in Arabidopsis demonstrates an essential role of saturated fatty acids in plant growth. Plant Cell 15, 1020–1033 (2003)

    Article  CAS  Google Scholar 

  21. DeLano, W. L. The PyMOL Molecular Graphics System. (DeLano Scientific, 2002)

  22. Collaborative Computational Project Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  23. Birney, E., Clamp, M. & Durbin, R. GeneWise and Genomewise. Genome Res. 14, 988–995 (2004)

    Article  CAS  Google Scholar 

  24. Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004)

    Article  CAS  Google Scholar 

  25. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007)

    Article  CAS  Google Scholar 

  26. Huelsenbeck, J. P. & Ronquist, F. MRBAYES: Bayesian inference of phylogeny. Bioinformatics 17, 754–755 (2001)

    Article  CAS  Google Scholar 

  27. Guindon, S., Lethiec, F., Duroux, P. & Gascuel, O. PHYML Online – a web server for fast maximum likelihood-based phylogenetic inference. Nucleic Acids Res. 33, W557–W559 (2005)

    Article  CAS  Google Scholar 

  28. Jez, J. M., Ferrer, J. L., Bowman, M. E., Dixon, R. A. & Noel, J. P. Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase. Biochemistry 39, 890–902 (2000)

    Article  CAS  Google Scholar 

  29. Doublié, S. Preparation of selenomethionyl proteins for phase determination. Methods Enzymol. 276, 523–530 (1997)

    Article  Google Scholar 

  30. Niesen, F. H., Berglund, H. & Vedadi, M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nature Protocols 9, 2212–2221 (2007)

    Article  Google Scholar 

  31. Leslie, A. G. W. Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography No. 26. (1992)

  32. Evans, P. R. Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2005)

    Article  Google Scholar 

  33. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  Google Scholar 

  34. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Cryst. 26, 795–800 (1993)

    Article  CAS  Google Scholar 

  35. Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849–861 (1999)

    Article  CAS  Google Scholar 

  36. Terwilliger, T. C. Automated main-chain model building by template matching and iterative fragment extension. Acta Crystallogr. D 59, 38–44 (2003)

    Article  Google Scholar 

  37. Vagin, A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Cryst. 30, 1022–1025 (1997)

    Article  CAS  Google Scholar 

  38. Sali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993)

    Article  CAS  Google Scholar 

  39. Perrakis, A., Morris, R. & Lamzin, V. S. Automated protein model building combined with iterative structure refinement. Nature Struct. Biol. 6, 458–463 (1999)

    Article  CAS  Google Scholar 

  40. Brunger, A. T. & Warren, G. L. Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  CAS  Google Scholar 

  41. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    Article  CAS  Google Scholar 

  42. McRee, D. E. XtalView/Xfit: a versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165 (1999)

    Article  CAS  Google Scholar 

  43. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  44. Collaborative Computational Project Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  45. Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D 60, 2256–2268 (2004)

    Article  CAS  Google Scholar 

  46. Invitrogen. Gateway technology: a universal technology to clone DNA sequences for functional analysis and expression in multiple systems. Catalog nos. 12535-019 and 12535-027. (2003)

  47. Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana . Plant J. 16, 735–743 (1998)

    Article  CAS  Google Scholar 

  48. Lohar, D. P., Shuller, K., Buzas, D. M., Gresshoff, P. M. & Stiller, J. Transformation of Lotus japonicum using the herbicide resistance bar gene as a selectable marker. J. Exp. Bot. 52, 1697–1702 (2001)

    Article  CAS  Google Scholar 

  49. Li, L., Ilarslan, H., James, M. G., Myers, A. M. & Wurtele, E. S. Genome wide co-expression among the starch debranching enzyme genes AtISA1, AtISA2, and AtISA3 in Arabidopsis thaliana . J. Exp. Bot. 58, 3323–3342 (2007)

    Article  CAS  Google Scholar 

  50. Earley, K. W. et al. Gateway-compatible vectors for plant functional genomics and proteomics. Plant J. 45, 616–629 (2006)

    Article  CAS  Google Scholar 

  51. Jefferson, R. A. Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5, 387–405 (1987)

    Article  CAS  Google Scholar 

  52. Doyle, J. J. & Doyle, J. L. Isolation of plant DNA from fresh tissue. Focus 12, 13–15 (1990)

    Google Scholar 

  53. Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Annu. Rev. Biochem. 72, 248–254 (1976)

    Article  CAS  Google Scholar 

  54. Markham, J. E., Li, J., Cahoon, E. B. & Jaworski, J. G. Separation and identification of major plant sphingolipid classes from leaves. J. Biol. Chem. 281, 22684–22694 (2006)

    Article  CAS  Google Scholar 

  55. Moon, S. & Nikolau, B. J. Plantmetabolomics Platform2: fatty acids. (2009)

  56. Perera, M. A. D. N., Dietrich, C. R., Meeley, R., Schnable, P. S. & Nikolau, B. J. in Advanced Research on Plant Lipids (eds Murata, M. et al.) 225–228 (Kluwer Academic, 2003)

    Book  Google Scholar 

  57. Fatland, B. L., Nikolau, B. J. & Wurtele, E. S. Reverse genetic characterization of cytosolic acetyl-CoA generation by ATP-citrate lyase in Arabidopsis . Plant Cell 17, 182–203 (2005)

    Article  CAS  Google Scholar 

  58. Davies, N. A. The New Automated Mass Spectrometry Deconvolution and Identification System (AMDIS) (ISAS, 1998)

    Google Scholar 

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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.

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Authors and Affiliations



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.

Corresponding authors

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).

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