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

Abstract

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.

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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.

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Acknowledgements

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).

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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.

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Correspondence to Hiroshi A Maeda.

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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)

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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). https://doi.org/10.1038/nchembio.2414

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