Article Figures & Data
Figures
- 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.
- 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.
- 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).
- 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).
- 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.
- 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).
Tables
Substrate kcat (s−1) Km (μM) kcat/Km (mM−1 s−1) C. tepidum PPA-AT homolog Prephenate 12.3 ± 0.2 464 ± 39 26.8 ± 2.5 α-Ketoglutarate 17.2 ± 1.9 949 ± 102 18.9 ± 3.9 Synechocystis sp PCC6803 PPA-AT homolog Prephenate 1.1 ± 0.1 2539 ± 254 0.44 ± 0.02 α-Ketoglutarate 54.2 ± 3.7 486 ± 33 111.7 ± 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.
Substrate kcat (s−1) Km (μM) kcat/Km (mM−1 s−1) Arogenate 5.4 ± 0.2 1544 ± 122 3.5 ± 0.2 Prephenate 0.27 ± 0.04 827 ± 87 0.32 ± 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).
Enzymes kcat (s−1) Km (μM) kcat/Km (mM−1 s−1) Prephenate (Asp amino donor) Wild type 21.3 ± 1.0 312 ± 30 69.0 ± 4.2 T84V 4.5 ± 1.2** 676 ± 96* 6.5 ± 0.9** K169S 5.5 ± 1.5** 791 ± 97* 6.7 ± 1.1** T84VK169S N.D. N.D. N.D. 4-Hydroxyphenylpyruvate (Trp amino donor) T84VK169S 5.0 ± 0.3 288 ± 15 17.2 ± 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).
Supplemental Data
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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
