Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants


L-Tyrosine (Tyr) is essential for protein synthesis and is a precursor of numerous specialized metabolites crucial for plant and human health. Tyr can be synthesized via two alternative routes by different key regulatory TyrA family enzymes, prephenate dehydrogenase (PDH, also known as TyrAp) or arogenate dehydrogenase (ADH, also known as TyrAa), representing a unique divergence of primary metabolic pathways. The molecular foundation underlying the evolution of these alternative Tyr pathways is currently unknown. Here we characterized recently diverged plant PDH and ADH enzymes, obtained the X-ray crystal structure of soybean PDH, and identified a single amino acid residue that defines TyrA substrate specificity and regulation. Structures of mutated PDHs co-crystallized with Tyr indicate that substitutions of Asn222 confer ADH activity and Tyr sensitivity. Reciprocal mutagenesis of the corresponding residue in divergent plant ADHs further introduced PDH activity and relaxed Tyr sensitivity, highlighting the critical role of this residue in TyrA substrate specificity that underlies the evolution of alternative Tyr biosynthetic pathways in plants.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Identification of noncanonical ADHs that are closely related to PDHs but have distinct substrate specificity and regulation.
Figure 2: X-ray crystal structure of soybean PDH1.
Figure 3: Identification of Asn222 as a determinant of PDH activity and Tyr sensitivity.
Figure 4: Crystal structures of GmPDH1 N222D and M219T N222D reveal Tyr binding interactions.
Figure 5: Asn222 confers PDH activity to divergent plant ADHs while simultaneously relaxing Tyr sensitivity.

Accession codes

Primary accessions

NCBI Reference Sequence

Protein Data Bank

Referenced accessions

Protein Data Bank


  1. 1

    Moghe, G.D. & Last, R.L. Something old, something new: conserved enzymes and the evolution of novelty in plant specialized metabolism. Plant Physiol. 169, 1512–1523 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Weng, J.-K., Philippe, R.N. & Noel, J.P. The rise of chemodiversity in plants. Science 336, 1667–1670 (2012).

    Article  CAS  Google Scholar 

  3. 3

    Gowik, U. & Westhoff, P. The path from C3 to C4 photosynthesis. Plant Physiol. 155, 56–63 (2011).

    Article  CAS  Google Scholar 

  4. 4

    Torruella, G., Suga, H., Riutort, M., Peretó, J. & Ruiz-Trillo, I. The evolutionary history of lysine biosynthesis pathways within eukaryotes. J. Mol. Evol. 69, 240–248 (2009).

    Article  CAS  Google Scholar 

  5. 5

    Jensen, R.A. & Pierson, D.L. Evolutionary implications of different types of microbial enzymology for L-tyrosine biosynthesis. Nature 254, 667–671 (1975).

    Article  CAS  Google Scholar 

  6. 6

    Schenck, C.A., Chen, S., Siehl, D.L. & Maeda, H.A. Non-plastidic, tyrosine-insensitive prephenate dehydrogenases from legumes. Nat. Chem. Biol. 11, 52–57 (2015).

    Article  CAS  Google Scholar 

  7. 7

    Maeda, H. & Dudareva, N. The shikimate pathway and aromatic amino acid biosynthesis in plants. Annu. Rev. Plant Biol. 63, 73–105 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Tzin, V. & Galili, G. New insights into the shikimate and aromatic amino acids biosynthesis pathways in plants. Mol. Plant 3, 956–972 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Fernstrom, J.D. & Fernstrom, M.H. Tyrosine, phenylalanine, and catecholamine synthesis and function in the brain. J. Nutr. 137 (Suppl. 1), 1539S–1547S (2007).

    Article  CAS  Google Scholar 

  10. 10

    Gleadow, R.M. & Møller, B.L. Cyanogenic glycosides: synthesis, physiology, and phenotypic plasticity. Annu. Rev. Plant Biol. 65, 155–185 (2014).

    Article  CAS  Google Scholar 

  11. 11

    Hagel, J.M. & Facchini, P.J. Benzylisoquinoline alkaloid metabolism: a century of discovery and a brave new world. Plant Cell Physiol. 54, 647–672 (2013).

    Article  CAS  Google Scholar 

  12. 12

    Hunter, S.C. & Cahoon, E.B. Enhancing vitamin E in oilseeds: unraveling tocopherol and tocotrienol biosynthesis. Lipids 42, 97–108 (2007).

    Article  CAS  Google Scholar 

  13. 13

    Millner, P.A. & Barber, J. Plastoquinone as a mobile redox carrier in the photosynthetic membrane. FEBS Lett. 169, 1–6 (1984).

    Article  CAS  Google Scholar 

  14. 14

    Strack, D., Vogt, T. & Schliemann, W. Recent advances in betalain research. Phytochemistry 62, 247–269 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Barros, J. et al. Role of bifunctional ammonia-lyase in grass cell wall biosynthesis. Nat. Plants 2, 16050 (2016).

    Article  CAS  Google Scholar 

  16. 16

    Bonner, C.A. et al. Cohesion group approach for evolutionary analysis of TyrA, a protein family with wide-ranging substrate specificities. Microbiol. Mol. Biol. Rev. 72, 13–53 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Fischer, R. & Jensen, R. Prephenate dehydrogenase (monofunctional). Methods Enzymol. 142, 503–507 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Hudson, G.S., Wong, V. & Davidson, B.E. Chorismate mutase/prephenate dehydrogenase from Escherichia coli K12: purification, characterization, and identification of a reactive cysteine. Biochemistry 23, 6240–6249 (1984).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Kuramitsu, S., Inoue, K., Ogawa, T., Ogawa, H. & Kagamiyama, H. Aromatic amino acid aminotransferase of Escherichia coli: nucleotide sequence of the tyrB gene. Biochem. Biophys. Res. Commun. 133, 134–139 (1985).

    Article  CAS  Google Scholar 

  20. 20

    Dal Cin, V. et al. Identification of genes in the phenylalanine metabolic pathway by ectopic expression of a MYB transcription factor in tomato fruit. Plant Cell 23, 2738–2753 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Dornfeld, C. et al. Phylobiochemical characterization of class-Ib aspartate/prephenate aminotransferases reveals evolution of the plant arogenate phenylalanine pathway. Plant Cell 26, 3101–3114 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Graindorge, M. et al. Identification of a plant gene encoding glutamate/aspartate-prephenate aminotransferase: the last homeless enzyme of aromatic amino acids biosynthesis. FEBS Lett. 584, 4357–4360 (2010).

    Article  CAS  Google Scholar 

  23. 23

    Maeda, H., Yoo, H. & Dudareva, N. Prephenate aminotransferase directs plant phenylalanine biosynthesis via arogenate. Nat. Chem. Biol. 7, 19–21 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Connelly, J.A. & Conn, E.E. Tyrosine biosynthesis in Sorghum bicolor: isolation and regulatory properties of arogenate dehydrogenase. Z. Naturforsch. C 41, 69–78 (1986).

    Article  CAS  Google Scholar 

  25. 25

    Gaines, C.G., Byng, G.S., Whitaker, R.J. & Jensen, R.A. L-tyrosine regulation and biosynthesis via arogenate dehydrogenase in suspension-cultured cells of Nicotiana silvestris Speg. et Comes. Planta 156, 233–240 (1982).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Rippert, P. & Matringe, M. Purification and kinetic analysis of the two recombinant arogenate dehydrogenase isoforms of Arabidopsis thaliana. Eur. J. Biochem. 269, 4753–4761 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Keller, B., Keller, E. & Lingens, F. Arogenate dehydrogenase from Streptomyces phaeochromogenes. Purification and properties. Biol. Chem. Hoppe Seyler 366, 1063–1066 (1985).

    Article  CAS  Google Scholar 

  28. 28

    Mayer, E., Waldner-Sander, S., Keller, B., Keller, E. & Lingens, F. Purification of arogenate dehydrogenase from Phenylobacterium immobile. FEBS Lett. 179, 208–212 (1985).

    Article  CAS  Google Scholar 

  29. 29

    Gamborg, O.L. & Keeley, F.W. Aromatic metabolism in plants. I. A study of the prephenate dehydrogenase from bean plants. Biochim. Biophys. Acta 115, 65–72 (1966).

    Article  CAS  Google Scholar 

  30. 30

    Rubin, J.L. & Jensen, R.A. Enzymology of L-tyrosine biosynthesis in mung bean (Vigna radiata [L.] Wilczek). Plant Physiol. 64, 727–734 (1979).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Legrand, P. et al. Biochemical characterization and crystal structure of Synechocystis arogenate dehydrogenase provide insights into catalytic reaction. Structure 14, 767–776 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Song, J., Bonner, C.A., Wolinsky, M. & Jensen, R.A. The TyrA family of aromatic-pathway dehydrogenases in phylogenetic context. BMC Biol. 3, 13 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Christendat, D., Saridakis, V.C. & Turnbull, J.L. Use of site-directed mutagenesis to identify residues specific for each reaction catalyzed by chorismate mutase-prephenate dehydrogenase from Escherichia coli. Biochemistry 37, 15703–15712 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Sun, W. et al. The crystal structure of Aquifex aeolicus prephenate dehydrogenase reveals the mode of tyrosine inhibition. J. Biol. Chem. 284, 13223–13232 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Christendat, D. & Turnbull, J.L. Identifying groups involved in the binding of prephenate to prephenate dehydrogenase from Escherichia coli. Biochemistry 38, 4782–4793 (1999).

    Article  CAS  Google Scholar 

  36. 36

    Sun, W., Singh, S., Zhang, R., Turnbull, J.L. & Christendat, D. Crystal structure of prephenate dehydrogenase from Aquifex aeolicus. Insights into the catalytic mechanism. J. Biol. Chem. 281, 12919–12928 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Lütke-Eversloh, T. & Stephanopoulos, G. Feedback inhibition of chorismate mutase/prephenate dehydrogenase (TyrA) of Escherichia coli: generation and characterization of tyrosine-insensitive mutants. Appl. Environ. Microbiol. 71, 7224–7228 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Chiu, H.J. et al. The structure of Haemophilus influenzae prephenate dehydrogenase suggests unique features of bifunctional TyrA enzymes. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 66, 1317–1325 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Ku, H., Park, S., Yang, I. & Kim, S. Expression and functional characterization of prephenate dehydrogenase from Streptococcus mutans. Process Biochem. 45, 607–612 (2010).

    Article  CAS  Google Scholar 

  40. 40

    Wierenga, R.K., De Maeyer, M.C.H. & Hol, W.G.J. Interaction of pyrophosphate moieties with alpha-helices in dinucleotide-binding proteins. Biochemistry 24, 1346–1357 (1985).

    Article  CAS  Google Scholar 

  41. 41

    Rippert, P., Puyaubert, J., Grisollet, D., Derrier, L. & Matringe, M. Tyrosine and phenylalanine are synthesized within the plastids in Arabidopsis. Plant Physiol. 149, 1251–1260 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Wang, M., Toda, K. & Maeda, H.A. Biochemical properties and subcellular localization of tyrosine aminotransferases in Arabidopsis thaliana. Phytochemistry 132, 16–25 (2016).

    Article  CAS  Google Scholar 

  43. 43

    Westfall, C.S., Xu, A. & Jez, J.M. Structural evolution of differential amino acid effector regulation in plant chorismate mutases. J. Biol. Chem. 289, 28619–28628 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Cardoso, D. et al. Revisiting the phylogeny of papilionoid legumes: new insights from comprehensively sampled early-branching lineages. Am. J. Bot. 99, 1991–2013 (2012).

    Article  Google Scholar 

  45. 45

    Wojciechowski, M.F., Lavin, M. & Sanderson, M.J. A phylogeny of legumes (Leguminosae) based on analysis of the plastid matK gene resolves many well-supported subclades within the family. Am. J. Bot. 91, 1846–1862 (2004).

    Article  CAS  Google Scholar 

  46. 46

    Reyes-Prieto, A. & Moustafa, A. Plastid-localized amino acid biosynthetic pathways of Plantae are predominantly composed of non-cyanobacterial enzymes. Sci. Rep. 2, 955–967 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Bonvin, J. et al. Biochemical characterization of prephenate dehydrogenase from the hyperthermophilic bacterium Aquifex aeolicus. Protein Sci. 15, 1417–1432 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Goodstein, D.M. et al. Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res. 40, D1178–D1186 (2012).

    Article  CAS  Google Scholar 

  49. 49

    Matasci, N. et al. Data access for the 1,000 Plants (1KP) project. Gigascience 3, 17 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  50. 50

    Dash, S. et al. Legume information system ( a key component of a set of federated data resources for the legume family. Nucleic Acids Res. 44, D1181–D1188 (2016).

  51. 51

    Fernández-Pozo, N. et al. EuroPineDB: a high-coverage web database for maritime pine transcriptome. BMC Genomics 12, 366 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  52. 52

    Whelan, S. & Goldman, N. A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach. Mol. Biol. Evol. 18, 691–699 (2001).

    CAS  Google Scholar 

  53. 53

    Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Saitou, N. & Nei, M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425 (1987).

    CAS  Google Scholar 

  56. 56

    Gorrec, F. The current approach to initial crystallization screening of proteins is under-sampled. J. Appl. Crystallogr. 46, 795–797 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. 57

    Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. HKL-3000: the integration of data reduction and structure solution–from diffraction images to an initial model in minutes. Acta Crystallogr. D Biol. Crystallogr. 62, 859–866 (2006).

    Article  CAS  Google Scholar 

  58. 58

    Sheldrick, G.M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008).

    Article  CAS  Google Scholar 

  59. 59

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

  60. 60

    Terwilliger, T.C. Maximum-likelihood density modification. Acta Crystallogr. D Biol. Crystallogr. 56, 965–972 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. 61

    Morris, R.J., Perrakis, A. & Lamzin, V.S. ARP/wARP and automatic interpretation of protein electron density maps. Methods Enzymol. 374, 229–244 (2003).

    Article  CAS  Google Scholar 

  62. 62

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

    Article  CAS  Google Scholar 

  63. 63

    Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 64

    McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. 65

    Maeda, H. et al. RNAi suppression of Arogenate Dehydratase1 reveals that phenylalanine is synthesized predominantly via the arogenate pathway in petunia petals. Plant Cell 22, 832–849 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. 66

    Trott, O. & Olson, A.J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 31, 455–461 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references


We thank K. Wallin (University of Minnesota) and U. (Rosia) Schmidt for the cloning of MtncADH and SlncADH, respectively, and P.-M. Delaux (Universite´ de Toulouse) for help with a legume phylogenetic analysis. This work was supported by the National Science Foundation (IOS-1354971 to H.A.M. and MCB-1614539 to J.M.J.). C.K.H. was supported by the NSF Graduate Research Fellowship Program (DGE-1143954). Portions of this research were carried out at the Argonne National Laboratory Structural Biology Center of the Advanced Photon Source, a national user facility operated by the University of Chicago for the Department of Energy Office of Biological and Environmental Research (DE-AC02-06CH11357).

Author information




C.A.S., C.K.H., J.M.J., and H.A.M. designed the research, C.A.S. performed phylogenetic analyses, and C.A.S., M.R.S., and Y.M. performed site-directed mutagenesis and biochemical characterization of recombinant enzymes. C.K.H. expressed, purified, and crystallized proteins, collected diffraction data, and determined crystal structures. S.G.L. assisted with SAD phasing, C.A.S., C.K.H., J.M.J., and H.A.M. analyzed data, and C.A.S., C.K.H., J.M.J., and H.A.M. wrote the paper with all authors providing editorial input.

Corresponding author

Correspondence to Hiroshi A Maeda.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–3, Supplementary Figures 1–8 (PDF 11055 kb)

Supplementary Dataset 1

Sequences used in the phylogenetic analyses from Figure 1 and Supplementary Figure 1 (XLSX 49 kb)

Supplementary Dataset 2

Full amino acid alignment made in MUSCLE used to construct the phylogenetic analyses in Supplementary Fig. 1 (TXT 278 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Schenck, C., Holland, C., Schneider, M. et al. Molecular basis of the evolution of alternative tyrosine biosynthetic routes in plants. Nat Chem Biol 13, 1029–1035 (2017).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing