a chorismate mutase (cm) - plant cell filea chorismate mutase (cm) b arogenate dehydrogenase (adh)...
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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
!
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 !
A.
PPA-A
T Chlorobi/Bacteroidetes PPA-AT Plantae
plants diatoms
fungi archaea
eubacteria cyanobacteria
alpha-proteobacteria chlorobi and bacteroidetes
!
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
!
(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
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
3.0
Clo_per_2
1_yre_ohR
Bra_sp._1
Fae_pra_1
Aci_sp._1
Thermus_th
ermophilus_
HB8
Lac_lac_1
1_nib_rtS
Psy_sp._1
Bac_myc_2
Myc_tub_1
Nei_sp._1
Met_bur_1
Ric_bel_1
Clo_bot_5
Geo_sp._2
Nit_sp._1
Moo_the_2
Hel_pyl_1
Aci_sac_1
Des_ole_1
Bur_am
b_1
Esc_col_1
Bif_lon_3
Fra_sp._
2
Clo_phy_1
Nit_sp._2
Ana_cac_1
Thi_cru_2
Pse_put_1
Geo_sp._1
Ana_col_1
Tur_sp._1
Oen_oen_1
Pop_tri_1
Aca_mar_1
Fus_sp._1
Met_the_1
Cam_lar_1
Clo_per_1
Bif_lon_1
Leu_mes_1
Ros_sp._1
Met_mar_1
Bac_ova_1
Deh_lyk_1
Thi_cru_1
Clo_asp_1Ph
o_pro_1
Bac_cer_12
1_alf
_leC
Bac_amy_1
Des_baa_2
1_nak_cyM
Sul_sp._1
Ric_can_1
Bif_lon_4
Bac_sp._2Fla_bac_1
Bif_bif_1
Ost_tau_1
AtAGD2
Ana_hyd_1
Str_gri_1Str_sca_1
Bac_cer_11
Des_des_1
Bac_cer_13
Pre_buc_1
Mes_lot_
1
Ast_exc_2
Bac_cer_8
Tro_whi_
1
Bif_bre_1
Tal_sti_1
Sul_del_1
Bac_xyl_1
Agr_rad_2
Hyd_sp._2
1_dad_ciD
del_pro_1
Rhi_leg_1
Aer_vir_1
Bac_pse_2
Lep_fer_1
Lac_del_1
Pol_irg_1
Ent_cas_1
Rho_sph_1
Cor_res_1
Rho_rub_1
Bac_cer_7
Rho_pal_2
Kor_alg_1
Str_ube_1
Azo_cau_1
Bac_vul_1
Pre_ori_1
Lok_ves_1
Des_mag_1
Lac_cas_2
Bif_cat_1
Syn_sp._1
Pro_stu_1
Kyt_sed_1
Des_sp._1
Cam_jej_1
Ped_sp._1
Nit_mob_2
Nat_pha_1
Bru_cet_1
Yer_ald_1
Bur_pse_1
Pse_aer_1
Com_tes_1
Sac_ery_1
AtCOR13
Bru_sui_2
Fer_pla_1
Noc_sp._1
Ver_spi_1
Ace_pas_1
Rho_pal_3
Ara_tha_4
Met_sp._1
Pyr_fur_1
Bru_sui_3
unc_bac_1
Met_ext_1
Pse_syr_1
Pse_atl_1
Aci_cal_1
The_afr_1
Bru_cet_3
Ali_den_1
Bac_cer_1
Str_coe_1
Ano_fla_1
Pep_ana_1
Ami_col_1
Ara_lyr_1
Fra_phi_1
Bac_sp._1Bur_vie_1
Bif_bif_2
Pre_mel_1
Bac_thu_1
Str_ora_1
Des_baa_
1
Pho_pro_2
Sel_spu_1
Gra_bet_1
At2G22250
Ana_var_1
Bac_dor_1Pet_mob_1
1_ic
a_il
A
Mar_aqu_1
Art_max_1
Met_eve_1
Pas_mul_1
Sal_ent_1
Met_pop_1
Bac_pse_1Lis_mon_2
Cat_mor_1
Clo_bot_2
gam_pro_2
She_pea_1
Clo_bot_
1
She_loi_1
1_oe
j_la
H
Bla_hyd_1
Zea_may_1
Hel_mod_1
Her_ser_1
The_mar_1
Bru_mel_1
Cit_sp._
1
Noc_far_1
Str_sp._1
Des_thi_1
Shu_sat_1
Bac_cer_3
The_sib_1
Can_Met_2
Bac_myc_1
Bru_sui_1
Mic_lut_1
Clo_nov_2
Pel_ber_1
Nei_sub_1
Des_pig_1
Lis_inn_1
The_kod_1
Bac_cer_4
Sal_rub_1
Str_aga_2
Her_ars_1
Rho_opa_1
Bur_pse_2
Clo_nov_1Clo_bot_4
1_da
r_ic
A
Str_sp._2
Met_tri_1
Nit_oce_1
Str_aga_1
Afi_sp._1
Fra_aln_1
Ped_sp._2
Sph_sp._1
Tri_ery_1
Kin_rad_
1
Lac_cas_1
Tre_vin_1
Mob_cur
_1
Rhi_sp._2
Yer_roh_1
Syn_sp._2
Sta_aur_1
Pyr_ars_
1
Ral_sol_1
Bac_ant_1
Dic_zea_1
Bul_ext_1
Bac_cer_2
Lee_bla_1
Sin_med_1
Nos_sp._1
Bur_amb_2
Ric_com_1
Sil_sp._1
unc_mar_1
Str_vir_1
Hyd_sp._1
Str_sal_1
Ery_lit_1
Can_tro
_1
Rho_m
ar_1
Shi_dys_1
Lis_mon_1
Met_nod_1
Aci_ave_1
Syn_aci_1
Rhi_sp._1
Eub_bif_1
Lac_cri_1
Rho_pal_1
Lis_see_1
Ric_aka_1
Ent_fae_1
Och_ant_1
Bac_uni_1
Erw_amy_1
Bac_sub_1
unc_eur_1
Geo_sp._3
Lep_fer_2
Str_sca_2
Lac_bre_1
Sid_lit_1
Bac_thu_2
Nit_mob_1
The_let_1
Bru_cet_2
Pse_flu_1
Des_fru_1
Bac_cer_6
Hae_som_1
Bac_cer_5
Fra_sp._1
Bac_cer_9
Oce_bat_1
Aqu_aeo_1
Par_joh_1
Eub_eli_1
Dor_lon_2
HsKota
Nos_pun_1
Chlorobium_tep
idum_TL
Hor_vul_1
Ato_rim_1
Clo_bot_3
Dan_rer_1
Agr_rad_1
Dor_lon_1
Bif_lon_2
Ast_exc_1
Ast_exc_3
Met_bar_1
Bac_cer_10
Moo_the_1
Ori_sin_1
Gra_ele_1
Alc_sp._1
gam_pro_1
Can_Met_1
Deh_sp._1
Met_rad_1
mar_gam
_1
Bur_sp._
1
Str_alb_1
Can_Met_3
The_pen_1
Bac_meg_1
100
100
86
100
100
77
100
100
100
08
93
100
100
100
100
100
100
100
100
93
54
100
100
100
100
100
100
79
99
100
92
100
100
100
100
64
100
100
81
100
100
100
65
100
71
90
100
99
100
100
001
100
100
73
100
100
100
100
001
78
100
100
100
100
99
100
100
100
100
100
100
100
100
100
100
100
100
60
100
100
100
97
100
100
100
100
99
100
100
69
100
100
100
83
100
100 100
100
96
100
100
100
100
75
100
100
100
100
100
100
100
54
100
80
100
100
80
100
95
93
100
100
76
100
100
100
77
100
64
100
100
100
100
10082
100
85
100
100
100
100
100
100
100
100
100
100
100
62
100
100
100
100
99
100
100
100
100
100
100
100
90
100
100
59
58
100
72
100
100
90
100
100
93
100
93
100
100
100
100
89
100
100
72
100
100
100
100
100
100
100
100
100
100100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
57
100
100
100
100
100
100
100
100
100
100
100
100
99
81
100
100
100
100
100
100
100
78
95
100
100100
100
100
100
100
100
100
100
100
100
100
65
100
100
65
80
80
100
100
100
001
81
100
98100
100
100
100
100
100
57
100
100
100
100
100
100
100
100
100
100
100
99
100
75
100
93
100
100
Thermus thermophilus (4)
Arabidopsis thaliana (1)
Chlorobium tepidum (3)
Synechocystis sp. PCC6803 (5)
Antirrhinum majus (2)
plants diatoms
fungi eubacteria
archaea α-proteobacteria
cyanobacteria chlorobi and bacteroidetes
Dickeya dadantii Ech703 (6)
Streptomyces bingchenggensis (7)
Alicyclobacillus acidocaldarius (8) Rhodothermus marinus_2
Clostridium perfringens
Functional PPA-AT homologs?
Rhodothermus marinus_1
Bacillus cereus G9842
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. !
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."
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.
!
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
Rhodothermus marinus_1 ZP_04423142.1 Rmar_0633 prephenate dehydrataseRmar_2129 Glu/Leu/Phe/Val dehydrogenaseRmar_1803 Prephenate dehydrogenase
Chloribium tepidum NP_661859.1 pheA prephenate dehydratasemdh malate dehydrogenasegdhA glutamate dehydrogenase
Thermus thermophilus YP_143312.1 ldh L-lactate dehydrogenaseTTHA1104 prephenate dehydratase ask aspartate kinase
Synechocystis 6803 NP_442191.1 mdh malate dehydrogenasepheA prephenate dehydratasegdhA glutamate dehydrogenase
Dickeya Dadantii YP_002987080.1 Dd703_1230 Glu/Leu/Phe/Val dehydrogenaseDd703_2853 chorismate mutaseDd703_3765 delta-1-pyrroline-5-carboxylate dehydrogenase
Alicyclobacillus acidocaldarius ZP_03494134.1 Aaci_2663 transcriptional regulator, AsnC familyAaci_1893 diaminopimelate epimerase Aaci_2662 protein of unknown function DUF322
Clostridium perfringens NP_562823.1 dapH tetrahydrodipicolinate succinylasemalA dipeptidase PepVCPE1908 cob(I)alamin adenosyltransferase
Bacillus cereus G9842 YP_002448463.1 BCG9842_B0200 transcriptional regulator, AsnC familyBCG9842_B0199 D-isomer specific 2-hydroxyacid dehydrogenase family proteincoaD phosphopantetheine adenylyltransferase
Rhodothermus marinus_2 YP_003290582.1 Rmar_0755 diaminopimelate epimeraseRmar_1657 dihydrodipicolinate reductaseRmar_1658 dihydrodipicolinate synthase
Supplemental Table 3. Aminotransferase activities of PPA-AT homologs with different keto acid acceptors shown in Figure 4B.
4-hydroxyprephenate α-ketoglutarate phenylpyruvate phenylpyruvate
(1) A. thaliana (AtPPA-AT) 295.4 ± 21.4 311.2 ± 9.1 N.D. N.D.(2) A. majus 308.9 ± 3.9 353.8 ± 5.2 N.D. N.D.(3) C. tepidum 241.7 ± 2.4 237.5 ± 6.1 N.D. N.D.(4) T. thermophilus 43.2 ± 8.6 79.9 ± 23.4 0.02 ± 0.01 N.D.(5) Synechocystis 6803 2.6 ± 0.0 202.1 ± 0.7 N.D. N.D.(6) D. dadantii 0.99 ± 0.01 45.5 ± 0.3 N.D. N.D.(7) S. bingchenggensis 0.14 ± 0.00 12.9 ± 0.1 0.65 ± 0.02 0.43 ± 0.08(8) A. acidocaldarius N.D. 4.5 ± 0.1 2.7 ± 0.1 0.65 ± 0.05
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)
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.
Aspartate Alanine Tryptophan
PPA-AT activityAtPPA-AT_WT 324.7 ± 29.0 0.29 ± 0.02 0.12 ± 0.02AtPPA-AT_T84V 66.9 ± 2.33 1.01 ± 0.08 0.89 ± 0.33AtPPA-AT_K169S 78.9 ± 4.38 1.18 ± 0.01 1.01 ± 0.36AtPPA-AT_T84VK169S N.D. N.D. N.D.
Asp-AT activityAtPPA-AT_WT 502.1 ± 10.6 N.D. N.D.AtPPA-AT_T84V 123.9 ± 7.6 0.87 ± 0.37 1.56 ± 0.59AtPPA-AT_K169S 145.1 ± 2.4 1.46 ± 0.11 1.84 ± 0.68AtPPA-AT_T84VK169S 0.81 ± 0.13 0.97 ± 0.03 1.05 ± 0.01
HPP-AT activityAtPPA-AT_WT N.D. 0.08 ± 0.01 N.D.AtPPA-AT_T84V 2.14 ± 0.30 0.66 ± 0.06 0.27 ± 0.07AtPPA-AT_K169S 2.30 ± 0.43 0.84 ± 0.05 0.30 ± 0.04AtPPA-AT_T84VK169S 0.36 ± 0.12 76.7 ± 11.2 123.3 ± 16.0
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)
Supplemental Table 5. Primer sequences used for cloning. A. majus PPA-AT homologs
Forward: 5’- CCATATGGCAGTATTGAAAACAGAGAAA -3’ Reverse: 5’- CGGATCCTTAGAGAGGAGCAGCAGGCT -3’
C. tepidum PPA-AT homologs
Forward: 5’- CACCATGAGCGTAGAGAGCTTTG -3’ Reverse: 5’- TTAACTGAACGCTTTTCTGATGC -3’
T. thermophilus PPA-AT homologs
Forward: 5’- CACCATGCGCGGCCTTTCCC -3’ Reverse: 5’- TTTCTAGGCGCGCCCCAG -3’
Synechocystis sp. PCC6803 PPA-AT homologs
Forward: 5’- CGCGCGGCAGCCATATGCGACTAACCCAGCGA -3’ Reverse: 5’- GACGGAGCTCGAATTCTCAAGCCAAAGTGCTGACAA -3’
D. dadantii Ech703 PPA-AT homologs
Forward: 5’- CGCGCGGCAGCCATATGAGAAGCGTAGCCGATC -3’ Reverse: 5’- GCTCGAATTCGGATCCTCATGATGCCTCCTGTTGC -3’
S. bingchenggensis PPA-AT homologs
Forward: 5’- CGCGCGGCAGCCATATGAATTCCACACTCGATCT -3’ Reverse: 5’- GACGGAGCTCGAATTCTCAGTTGCTTTGCACGGT -3’
A. acidocaldarius PPA-AT homologs
Forward: 5’-CGCGCGGCAGCCATATGAACACGCTGTATGACC-3’ Reverse: 5’-GACGGAGCTCGAATTCCTATCTCACCGGGTGATAC-3’
C. tepidum ADT homologs Forward: 5’- CACCATGACAAACTGGTTGATCG -3’ Reverse: 5’- TCGTCATGGATTCACCACCC -3’
Supplemental References:
Bonner CA, Jensen RA (1985) Novel features of prephenate aminotransferase from cell cultures
of Nicotiana silvestris. Arch. Biochem. Biophys. 238: 237–246
Bonner CA, Jensen RA (1987) A selective assay for prephenate aminotransferase activity in
suspension-cultured cells of Nicotiana silvestris. Planta 172: 417–423
De-Eknamkul W, Ellis BE (1988) Purification and characterization of prephenate
aminotransferase from Anchusa officinalis cell cultures. Arch. Biochem. Biophys. 267: 87–
94
Nakai T, Okada K, Akutsu S, Miyahara I, Kawaguchi S, Kato R, Kuramitsu S, Hirotsu K
(1999) Structure of Thermus thermophilus HB8 aspartate aminotransferase and its complex
with maleate. Biochemistry (Mosc) 38: 2413–2424
Niesen FH, Berglund H, Vedadi M (2007) The use of differential scanning fluorimetry to detect
ligand interactions that promote protein stability. Nat. Protoc. 2: 2212–2221
Siehl DL, Connelly JA, Conn EE (1986) Tyrosine biosynthesis in Sorghum bicolor:
characteristics of prephenate aminotransferase. Z. Naturforschung C- J Biosci 41: 79–86
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