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Research ArticleResearch Article
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Phylobiochemical Characterization of Class-Ib Aspartate/Prephenate Aminotransferases Reveals Evolution of the Plant Arogenate Phenylalanine Pathway

Camilla Dornfeld, Alexandra J. Weisberg, Ritesh K C, Natalia Dudareva, John G. Jelesko, Hiroshi A. Maeda
Camilla Dornfeld
Department of Botany, University of Wisconsin-Madison, Madison, Wisconsin 53706
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Alexandra J. Weisberg
Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061
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Ritesh K C
Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061
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Natalia Dudareva
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907
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John G. Jelesko
Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061
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Hiroshi A. Maeda
Department of Botany, University of Wisconsin-Madison, Madison, Wisconsin 53706
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  • ORCID record for Hiroshi A. Maeda
  • For correspondence: maeda2@wisc.edu

Published July 2014. DOI: https://doi.org/10.1105/tpc.114.127407

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    Figure 1.

    Different Pathways for Phe and Tyr Biosynthesis.

    Phe and Tyr can each be synthesized from prephenate via two alternative routes, the arogenate pathway and/or the phenylpyruvate/4-hydroxyphenylpyruvate pathways. ADH (EC 1.3.1.79); ADT (EC 4.2.1.91); CM (EC 5.4.99.5); HPP-AT, 4-hydroxyphenylpyruvate aminotransferase; PDH (EC 1.3.1.12); PDT (EC 4.2.1.51); PPA-AT (EC 2.6.1.79); PPY-AT, phenylpyruvate aminotransferase.

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    Figure 2.

    ML Phylogenetic Analyses of PPA-AT and ADT.

    Node labels are nonparametric bootstrap percentage values. Colored bars and labels over phylograms indicate monophyletic groups. Trees for PPA-AT (A) and ADT (B) are arbitrarily rooted on the plant homolog used in the similarity searches.

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    Figure 3.

    PPA-AT, AspAT, PPY-AT, and HPP-AT Activity Assays Using C. tepidum PPA-AT Homolog.

    (A) Arogenate (Agn) was produced after 15 min incubation of the recombinant C. tepidum enzyme (20 μg/mL) with 1 mM prephenate substrate, 5 mM aspartate (Asp) amino donor, and 200 μM PLP cofactor at 37°C (solid line). The reactions lacking prephenate, Asp, or the enzyme did not produce Agn (dotted lines).

    (B) The C. tepidum enzyme converted α-ketoglutarate into glutamate (Asp-AT activity) but did not use phenylpyruvate (PPY-AT activity) and 4-hydroxyphenylpyruvate (HPP-AT activity).

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    Figure 4.

    Aminotransferase Activity of PPA-AT Homologs from Different Organisms.

    (A) Significant arogenate production was observed in PPA-AT reactions containing (1) Arabidopsis, (2) A. majus, (3) C. tepidum, (4) T. thermophilus, (5) Synechocystis sp PCC6803, or (6) D. dadantii enzymes, but not with (7) S. bingchenggensis, (8) A. acidocaldarius, or (0) no enzyme control, after incubation with 1 mM prephenate, 5 mM l-aspartate, and 200 μM PLP for 35 min at 37°C.

    (B) The activity of PPA-AT, Asp-AT, PPY-AT, and HPP-AT for individual enzymes was measured after incubation with 1 mM keto acid substrate, 5 mM l-aspartate, and 200 μM PLP for 15 min at 37°C. N.D., below detection limit. Data are means ± se (n ≥ 3).

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    Figure 5.

    Identification of Thr-84 and Lys-169 as Highly Conserved among AspAT Ib Enzymes Having PPA-AT Activity.

    Primary peptide sequence alignment of prephenate utilizing and not utilizing PPA-AT homologs (above and below the dotted line) revealed Thr-84 and Lys-169 are highly conserved only among AspAT Ib enzymes exhibiting PPA-AT activity. Phylogenetic relationships of individual enzymes are shown in Supplemental Figure 6, except for PPA-AT homologs of Petunia hybrida (HM635905.1), Salinibacter ruber (YP_445071.1), and Ostreococcus lucimarinus (XP_001421566.1; Figure 2). Alignments were performed by ClustalW with default settings and shaded using the BoxShade Version 3.21 software program.

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    Figure 6.

    Amino Donor and Keto Acceptor Preference of Arabidopsis PPA-AT Wild Type and Mutants.

    PPA-AT (A), Asp-AT (B), and HPP-AT (C) activities were analyzed in the wild type, T84V and K169S single, and T84VK169S double mutants using l-aspartate (Asp), l-alanine (Ala), or l-tryptophan (Trp) as an amino donor. The structures of respective keto acid substrates, prephenate (A), α-ketoglutarate (B), and 4-hydroxyphenylpyruvate (C), are shown in each panel. The reaction mixtures containing 3 mM keto acid substrate, 20 mM amino donor, 200 μM PLP, and 0.5 to 20 μg/mL purified recombinant enzyme were incubated at 37°C for 10 min. Data are means of three independent experiments, and individual standard errors are shown in Supplemental Table 4. Open squares: activity was not detectable (Supplemental Table 4).

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    Table 1. Kinetic Analysis of C. tepidum and Synechocystis sp PCC6803 PPA-AT Homologs
    Substratekcat (s−1)Km (μM)kcat/Km (mM−1 s−1)
    C. tepidum PPA-AT homolog
     Prephenate12.3 ± 0.2464 ± 3926.8 ± 2.5
     α-Ketoglutarate17.2 ± 1.9949 ± 10218.9 ± 3.9
    Synechocystis sp PCC6803 PPA-AT homolog
     Prephenate1.1 ± 0.12539 ± 2540.44 ± 0.02
     α-Ketoglutarate54.2 ± 3.7486 ± 33111.7 ± 5.7
    • Kinetic parameters were obtained for prephenate and α-ketoglutarate in 5-min reactions using 0.5 and 2 μg/mL recombinant enzymes, respectively, for C. tepidum PPA-AT homolog and 8 and 0.5 μg/mL for Synechocystis homolog. Data are means ± se (n = 3 independent experiments). 20 mM aspartate and 200 μM PLP were used as an amino donor and a cofactor, respectively.

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    Table 2. Kinetic Analysis of PDT and ADT Activity of the C. tepidum ADT Homolog
    Substratekcat (s−1)Km (μM)kcat/Km (mM−1 s−1)
    Arogenate5.4 ± 0.21544 ± 1223.5 ± 0.2
    Prephenate0.27 ± 0.04827 ± 870.32 ± 0.02
    • Kinetic parameters were obtained for arogenate (ADT activity) and prephenate (PDT activity) in 5- and 30-min reactions using 2 and 37.2 μg/mL recombinant enzyme, respectively. Data are means ± se (n = 3 independent experiments).

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    Table 3. Kinetic Analysis of Arabidopsis PPA-AT Wild Type and Mutants
    Enzymeskcat (s−1)Km (μM)kcat/Km (mM−1 s−1)
    Prephenate (Asp amino donor)
    Wild type21.3 ± 1.0312 ± 3069.0 ± 4.2
    T84V4.5 ± 1.2**676 ± 96*6.5 ± 0.9**
    K169S5.5 ± 1.5**791 ± 97*6.7 ± 1.1**
    T84VK169SN.D.N.D.N.D.
    4-Hydroxyphenylpyruvate (Trp amino donor)
    T84VK169S5.0 ± 0.3288 ± 1517.2 ± 0.7
    • Kinetic parameters were obtained for prephenate using aspartate amino donor (20 mM) in 10-min reactions using 0.5 and 2 μg/mL recombinant enzymes for wild type and mutants of Arabidopsis PPA-AT enzymes, respectively. For the T84VK169S double mutant, PPA-AT activity was not detectable (N.D.); instead, kinetic parameters were obtained for 4-hydroxyphenylpyruvate using Trp amino donor (20 mM) in 3-min reactions using 4 μg/mL recombinant enzyme. PLP (200 μM) was used as a cofactor. Data are means ± se (n ≥ 3). Significant difference from the corresponding wild type value is indicated (*P < 0.05, **P < 0.01, Student’s t test).

Additional Files

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  • Supplemental Data

    Files in this Data Supplement:

    • Supplemental Figures and Tables
    • Supplemental Dataset 3
    • Supplemental Dataset 1
    • Supplemental Dataset 2
    • Supplemental Dataset 4
  • Correction

    A correction has been published for this article:

    Dornfeld et al. (2014). Phylobiochemical Characterization of Prephenate Aminotransferases Reveals Evolution of the Plant Arogenate Phenylalanine Pathway. Plant Cell. 26:3101-3114 Correction: http://dx.doi.org/10.1105/tpc.16.00951

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    Phylobiochemical Characterization of Class-Ib Aspartate/Prephenate Aminotransferases Reveals Evolution of the Plant Arogenate Phenylalanine Pathway
    Camilla Dornfeld, Alexandra J. Weisberg, Ritesh K C, Natalia Dudareva, John G. Jelesko, Hiroshi A. Maeda
    The Plant Cell Jul 2014, 26 (7) 3101-3114; DOI: 10.1105/tpc.114.127407

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    Phylobiochemical Characterization of Class-Ib Aspartate/Prephenate Aminotransferases Reveals Evolution of the Plant Arogenate Phenylalanine Pathway
    Camilla Dornfeld, Alexandra J. Weisberg, Ritesh K C, Natalia Dudareva, John G. Jelesko, Hiroshi A. Maeda
    The Plant Cell Jul 2014, 26 (7) 3101-3114; DOI: 10.1105/tpc.114.127407
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