A Chorismate mutase (CM) B Arogenate dehydrogenase (ADH) Supplemental Figure 1. ML phylogenetic non-parametric bootstrap consensus analyses of CM and ADH. Trees are unrooted with arbitrary branch lengths; node labels are given as bootstrap support percentages. Taxa are colored coded as follows: pink, archaea; black, assorted eubacteria; red, diatoms; green, Plantae; teal, cyanobacteria; brown, α-proteobacteria; and orange, Bacteroidetes or Chlorobi. plants diatoms fungi archaea eubacteria cyanobacteria alpha-proteobacteria chlorobi and bacteroidetes
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A Chorismate mutase (CM) - Plant Cell fileA Chorismate mutase (CM) B Arogenate dehydrogenase (ADH) Supplemental Figure 1. ML phylogenetic non-parametric bootstrap consensus analyses
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A Chorismate mutase (CM)
B Arogenate dehydrogenase (ADH)
Supplemental Figure 1. ML phylogenetic non-parametric bootstrap consensus analyses of CM and ADH. Trees are unrooted with arbitrary branch lengths; node labels are given as bootstrap support percentages. Taxa are colored coded as follows: pink, archaea; black, assorted eubacteria; red, diatoms; green, Plantae; teal, cyanobacteria; brown, α-proteobacteria; and orange, Bacteroidetes or Chlorobi.
plants diatoms
fungi archaea
eubacteria cyanobacteria
alpha-proteobacteria chlorobi and bacteroidetes
!
Hiroshi A. Maeda
Hiroshi A. Maeda
Supplemental Data. Dornfeld et al. (2014). Plant Cell 10.1105/tpc.114.127407
Hiroshi A. Maeda
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Hiroshi A. Maeda
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Supplemental Figure 2. PPA-AT optimal ML search trees showing estimated branch lengths. Non-parametric bootstrap support values (out of 1024 replicates) are mapped onto nodes. PPA-AT-specific clades are indicated by colored bars over relevant taxa. Taxon color-coding is the same as Figure 2 and Supplemental Figure 1 online.
PPA-AT Plantae
PPA-AT Bacteroidetes/Chlorobiplants
diatoms fungi
archaea eubacteria
cyanobacteria alpha-proteobacteria
chlorobi and bacteroidetes !
Hiroshi A. Maeda
Hiroshi A. Maeda
Supplemental Data. Dornfeld et al. (2014). Plant Cell 10.1105/tpc.114.127407
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A.
PPA-A
T Chlorobi/Bacteroidetes PPA-AT Plantae
plants diatoms
fungi archaea
eubacteria cyanobacteria
alpha-proteobacteria chlorobi and bacteroidetes
!
Hiroshi A. Maeda
Supplemental Data. Dornfeld et al. (2014). Plant Cell 10.1105/tpc.114.127407
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B.
Supplemental Figure 3. PPA-AT and ADT optimal ML search trees used for the approximately unbiased (AU) test shown in Supplemental Table 1. Estimated branch lengths are shown. Non-parametric bootstrap support values (out of 1024 replicates each) are mapped onto nodes. Taxon color-coding is the same as Figure 2 and Supplemental Figure 1 online. (A) Unconstrained PPA-AT ML phylogram. (B) Unconstrained ADT ML phylogram.
plants diatoms
fungi archaea
eubacteria cyanobacteria
alpha-proteobacteria chlorobi and bacteroidetes
!
Hiroshi A. Maeda
Supplemental Data. Dornfeld et al. (2014). Plant Cell 10.1105/tpc.114.127407
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(kDa)
70 55 40 35 25
15
130
T84K
169
K16
9
T84
WT
A. m
ajus
T. th
erm
ophi
lus
D. d
adan
tii
S. b
ingc
heng
gens
is
A. a
cido
cald
ariu
s
A
B
(kDa)
70 55 40 35 25
15
130
(kDa)
70 55 40 35 25
15
130
C. tepidum PPA-AT homolog
C
Supplemental Figure 4. SDS-PAGE of purified recombinant enzymes. (A) E. coli protein crude extracts expressing C. tepidum and Synechocystis PPA-AT homologs and A. thaliana PPA-AT (Lane 2) were purified to 47.2, 44.5, and 47.7 kDa proteins, respectively (Lane 3). Lane 1 represents E. coli extracts expressing an empty vector. (B) Purified recombinant proteins of the T84VK169S double mutant, the K169S and T84V single mutants, wild type of A. thaliana PPA-AT (47.7 kDa), as well as PPA-AT homologs of A. majus (47.3 kDa), T. thermophilus (45.2 kDa), D. dadantii (46.4 kDa), S. bingchenggensis (45.2 kDa), and A. acidocaldarius (45.5 kDa). (C) E. coli protein crude extract expressing C. tepidum ADT homolog (Lane 1) was purified to 34.3 kDa proteins (Lane 5). Most of the expressed C. tepidum ADT homolog was found in the pellet (Lane 3).!
1 2 3
Synechocystis PPA-AT homolog
A. thaliana PPA-AT 1 2 3
C. tepidum ADT homolog
A. thaliana PPA-AT
(kDa)
70 55 40 35 25
15
130
1 2 3
(kDa)
70 55 40
35 25
15
130
1. C
tAD
T cr
ude
2. E
mpt
y cr
ude
3. C
tAD
T pe
llet
4. E
mpt
y pe
llet
5. P
urifi
ed C
tAD
T
Hiroshi A. Maeda
Supplemental Data. Dornfeld et al. (2014). Plant Cell 10.1105/tpc.114.127407
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Supplemental Figure 5. Time-dependent ADT and PDT activity of C. tepidum ADT homolog. !ADT and PDT activity of the purified recombinant C. tepidum enzyme were measured with indicated reaction times. For ADT activity, 0.5 mM arogenate substrate was incubated at 37°C with 4 μg/mL enzyme and the production of Phe was analyzed. For PDT activity, 1 mM prephenate substrate was incubated at 37°C with 37.2 μg/mL enzyme and the production of phenylpyruvate was measured. !!
0
3
6
9
12
15
0 15 30 45 60
Pro
duct
form
atio
n (µ
mol
/mg
prot
ein)
Reaction time (min)
PDT activity
ADT activity
Hiroshi A. Maeda
Supplemental Data. Dornfeld et al. (2014). Plant Cell 10.1105/tpc.114.127407
Supplemental Figure 6. !Prediction of the transition between functional and non-functional PPA-ATs using a deep ML phylogenetic tree. The tree is unrooted with arbitrary branch lengths; node labels are given as ML consensus values. Taxa are colored coded as Figure 2. Representative PPA-AT homologs used for the recombinant enzyme characterization (numbered from one to eight) and/or the functional protein-protein association analysis (Supplemental Table 2) are indicated at the locations where they are found or can be placed (i.e., Rhodothermus marinus_1 enzyme was placed between plant and C. tepidum enzymes based on the results from Figure 2). A group of potentially functional PPA-AT homologs (based on the analysis on Supplemental Table 2) is indicated by blue arrows. !
Hiroshi A. Maeda
Supplemental Data. Dornfeld et al. (2014). Plant Cell 10.1105/tpc.114.127407
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AtPPA-AT wild type
AtPPA-AT_T84V mutant
Temperature (°C)
AtPPA-AT_K169S mutant
AtPPA-AT_T84V_K169S mutant
No enzyme control
81.6 ± 0.4°C
83.1 ± 0.2°C**
82.1 ± 0.1°C
83.0 ± 0.3°C*
Supplemental Figure 7. !Representative thermo-denaturation responses of wild type and mutants of Arabidopsis PPA-AT (AtPPA-AT) enzymes. Melting temperatures (Tm) of the purified recombinant enzymes were estimated by differential scanning fluorimetry (DSF). The individual enzymes were mixed with the fluorescence dye, SYPRO orange, and the fluorescence signal was monitored during step wise increase in temperature (1°C per minute from 25 to 99°C) (Niesen et al., 2007). The Tm was calculated by non-linear regression analysis using the Boltzmann Sigmoidal equation and shown as means ± S.E. (n ≥ 3). Significantly different from the corresponding wild type value (*P<0.05, **P<0.01, Student-t test). Relatively high Tm values of the AtPPA-AT enzymes are consistent with the thermostable properties of PPA-AT activities detected in previous biochemical studies (Bonner and Jensen, 1985; Siehl et al., 1986; Bonner and Jensen, 1987; De-Eknamkul and Ellis, 1988) and are currently under investigation."
Hiroshi A. Maeda
Supplemental Data. Dornfeld et al. (2014). Plant Cell 10.1105/tpc.114.127407
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Supplemental Table 1.
Approximately unbiased (AU) test of ADT and PPA-AT phylogenies.
Dataset p-value PPA-AT unconstrained (Plantae PPA-AT group with bacteroidetes/chlorobi)
1.00
Plantae PPA-AT constrained with α-proteobacteria 4e-05 Plantae PPA-AT constrained with cyanobacteria 3e-08 ADT unconstrained (Plantae ADT group with bacteroidetes/chlorobi)
1.00
Plantae ADT constrained with α-proteobacteria 1e-05 Plantae ADT constrained with cyanobacteria 4e-52
The approximately unbiased (AU) test creates a distribution based on pseudo-replicates generated from the site-wise log-likelihood scores for all of the trees in the test using the multi-scale bootstrap technique (1). A p-value for each tree is then calculated based on the distribution. Trees that are rejected (p-value < 0.05) are significantly worse than trees that are not. This table contains the results of hypothesis testing of different evolutionary hypotheses for PPA-AT and ADT. In this case, the tree with the largest lnL for each hypothesis (out of 1024 search replicates each) was selected for AU testing using Consel (2). In both PPA-AT and ADT, the unconstrained trees (Supplemental Figure 3A and B online, where plants and algae clustered closest to the chlorobi/bacteroidetes) were significantly better than those clustered with either cyanobacteria or α-proteobacteria. These results provide support for PPA-AT and ADT having a chlorobi/bacteroidetes origin rather than being more likely originated from cyanobacteria or α-proteobacteria. 1. Shimodaira H (2002) An approximately unbiased test of phylogenetic tree selection. Syst Biol 51:492–
508.
2. Shimodaira H, Hasegawa M (2001) CONSEL: for assessing the confidence of phylogenetic tree selection. Bioinforma 17:1246–1247.
!
Hiroshi A. Maeda
Supplemental Data. Dornfeld et al. (2014). Plant Cell 10.1105/tpc.114.127407
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Supplemental Table 2. Functional protein association prediction of PPA-AT homologs. Top three candidate proteins that are functionally associated with potential PPA-AT homologs were identified by STRING (http://string-db.org/). Proteins involved in Phe or Tyr biosynthesis are shown in blue. The dotted blue line denotes a predicted transition between functional and non-functional PPA-ATs.
Organism Query protein Predicted functional partners
Arabidopsis thaliana NP_565529.1 AT5G57850 aminotransferase class IV family proteinAT3G53580 diaminopimelate epimerase family proteinAT5G13520 peptidase M1 family protein
Activities were measured in 15 min reactions at 37°C using 1 mM keto acid substrate, 5 mM aspartate amino donor, and 200 µM PLP cofactor.Different enzyme concentrations were used, so that activity was proportional to enzyme concentration.Activities (nmol s-1 mg protein-1) are shown as means ± S.E (n ≥ 3).N.D., Not detectable or below detection limit (< 0.01 nmol s-1 mg protein-1)
Hiroshi A. Maeda
Supplemental Data. Dornfeld et al. (2014). Plant Cell 10.1105/tpc.114.127407
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Supplemental Table 4. PPA-AT, Asp-AT, and HPP-AT activities of A. thaliana PPA-AT wild type and mutants with different amino donors shown in Figure 6.
Activities were measured in 10 min reactions at 37°C using 3 mM keto acid substrate, 20 mM aspartateamino donor, and 200 µM PLP cofactor. Different enzyme concentrations (0.5 to 20 µg/mL) were used, so that activity was proportional to enzyme concentration.Activities (nmol s-1 mg protein-1) are shown as means ± S.E (n = 3).N.D., Not detectable or below detection limit (< 0.01 nmol s-1 mg protein-1)
Hiroshi A. Maeda
Supplemental Data. Dornfeld et al. (2014). Plant Cell 10.1105/tpc.114.127407
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Supplemental Table 5. Primer sequences used for cloning. A. majus PPA-AT homologs