Purification and kinetic analysis of the two recombinant arogenatePascal Rippert and Michel Matringe Laboratoire Mixte CNRS/INRA/Bayer CropScience UMR 1932, Lyon, France The present stud
Trang 1Purification and kinetic analysis of the two recombinant arogenate
Pascal Rippert and Michel Matringe
Laboratoire Mixte CNRS/INRA/Bayer CropScience (UMR 1932), Lyon, France
The present study reports the first purification and kinetic
characterization of two plant arogenate dehydrogenases
(EC 1.3.1.43), an enzyme that catalyses the oxidative
decarboxylation of arogenate into tyrosine in presence of
NADP The two Arabidopsis thaliana arogenate
dehy-drogenases TyrAAT1 and TyrAAT2 were overproduced
in Escherichia coli and purified to homogeneity
Bio-chemical comparison of the two forms revealed that at
low substrate concentration TyrAAT1 is four times more
efficient in catalyzing the arogenate dehydrogenase
reac-tion than TyrAAT2 Moreover, TyrAAT2 presents a
weak prephenate dehydrogenase activity whereas Tyr-AAT1 does not The mechanism of the dehydrogenase reaction catalyzed by these two forms has been investi-gated using steady-state kinetics For both enzymes, steady-state velocity patterns are consistent with a rapid equilibrium, random mechanism in which two dead-end complexes, E–NADPH–arogenate and E–NADP–tyro-sine, are formed
Keywords: Arabidopsis thaliana; tyrosine biosynthesis; arogenate dehydrogenase; isoforms; enzymatic properties
Archae, eubacteria, plants, and fungi are capable of
synthesizing, de novo, the three aromatic amino acids
phenylalanine, tyrosine, and tryptophan The enzymes
catalyzing these reactions are thus potentially useful targets
for the development of new antibiotics, fungicides and
herbicides The broad-spectrumherbicide glyphosate that
inhibits 5-enolpyruvyl shikimate 3-phosphate synthase is
the best example of this potential [1]
For the biosynthesis of phenylalanine and tyrosine,
alternative routes exist in nature (Fig 1) In organisms
such as Saccharomyces cerevisiae or Escherichia coli [2,3],
phenylpyruvate and p-hydroxyphenylpyruvate serve as
the direct precursors of phenylalanine and tyrosine,
respectively In most plant however, both amino acids
are formed from a common precursorL-arogenate [4–7]
Furthermore, a widespread combination of the
alternat-ive routes can be found For example, in cyanobacteria,
and some other microorganisms, both
arogenate-to-tyrosine and phenylpyruvate-to-phenylalanine pathways
exist [8–12] In other bacteria, e.g Pseudomonas
aerugi-nosaand Zymomonas mobilis, the alternative pathways to
phenylalanine and tyrosine coexist [13,14] Whatever the
synthetic route adopted, a fine tuning at the branch
points is required to balance the flow of intermediates; a
wide variety of control mechanisms have been reported,
including multivalent allosteric feed back control by end products (reviewed in [15–17]) In most organisms, the aromatic amino acid biosynthetic pathway also plays a pivotal role in the production of precursors for a myriad
of aromatic secondary metabolites engaged in a very diverse range of processes [18] This opens the possibility
to deregulate the pathway in favor of one of the three aromatic amino acids, by overexpressing a foreign enzyme with a completely different pattern of regulation However, the existence of many different combinations for routing prephenate to phenylalanine or tyrosine means that the substrate specificity of a particular enzyme, and its susceptibility to feedback regulation by different metabolites is not predictable, and must be studied in detail
In a previous study [19], we reported on the identi-fication of two structural genes encoding plastidic Arabidopsis thaliana arogenate dehydrogenase named, respectively, tyrAAT1 (accession number AF434681), and tyrAAT2 (accession number AF434682), and the characterization of the recombinant protein TyrAAT1 This protein was structurally unusual in that a single polypeptide chain housed two highly similar domains Our study revealed that both peptide domains sustained arogenate dehydrogenase activity with similar biochemi-cal characteristics The second isoform, TyrAAT2, appeared later in the A thaliana databases [20] and did not present these repeated peptide domains The aim of the present study was to conduct a biochemical compar-ison of these two isoforms of arogenate dehydro-genase and a detailed kinetic analysis of their substrate specificity and reaction mechanisms to gain further insight into their respective roles in tyrosine metabolism
To date, reports of such mechanistic studies have been limited to a related bifunctional enzyme choris-mate mutase-prephenate dehydrogenase from bacteria [3,21,22]
Correspondence to M Matringe, Laboratoire Mixte CNRS/INRA/
Bayer CropScience (UMR 1932), Bayer CropScience,
14–20 rue Pierre Baizet, 69263 Lyon cedex 9, France.
Fax: + 33 472 85 22 97, Tel.: + 33 472 85 28 47,
E-mail: michel.matringe@bayercropscience.com
Abbreviations: TyrAAT1, Arabidopsis thaliana arogenate
dehydrogenase isoform1; TyrAAT2, Arabidopsis thaliana
arogenate dehydrogenase isoform2.
(Received 7 June 2002, revised 2 August 2002,
accepted 6 August 2002)
Trang 2M A T E R I A L S A N D M E T H O D S
Materials
Isopropyl thio-b-D-galactoside was purchased
fromBio-probe Prephenate, NAD, NADP, benzamidine HCl, amino
caproic acid were obtained fromSigma
Arogenate was synthesized enzymatically from
prephen-ate and aspartprephen-ate as described by Rippert & Matringe
[19] Arogenate was further purified fromthe reaction using
the following steps as described by Jensen et al [28]
The arogenate solution in 1 mM potassiumphosphate,
pH 8.0 was passed through a Dowex-chloride column
(1.6· 30 cm, Sigma) at a flow rate of 2 mLÆmin)1 The
column was then washed with 100 mL of 1 mMpotassium
phosphate, pH 8.0 Arogenate is eluted fromthe
Dowex-chloride column with a 100 mL of 0.5M NH4HCO3
Arogenate eluted was then lyophilized, resuspended in
50 mMHepps pH 8.6 and stored frozen at)20 C
Bacterial strains
The E coli AT 2471 [23], tyrA relA spoT thi mutant was
purchased from E coli Genetic Stock Center This mutant
was lysogenized with the helper phage (kDE3) harboring a
copy of the T7 RNA polymerase gene, using the kDE3
lysogenization kit fromNovagen, according to
manufac-turer’s instructions The resulting E coli AT 2471(kDE3)
was used to express cDNAs cloned in the pET vector under
the control of the T7 promoter Electro-competent cells of
this strain, prepared according to the method of Dower
et al [24], were transformed with the pET28-TyrAAT1 and
pET21-TyrAAT2 constructs
Engineering of expression vector BLAST search using available prephenate and arogenate dehydrogenase protein sequences allowed us to identify two
A thalianaarogenate dehydrogenase genes (accession num-ber AF434681 and AF434682) [19] The corresponding full-length cDNAs were obtained by PCR amplification of an Arabidopsis(var Columbia) cDNA library constructed in pYes The cloning of tyrAAT1 was previously described [19] under the name tyrAATc The NdeI–BamHI DNA frag-ments containing the coding sequence without the transit peptide [19] was cloned into the plasmid pET28 b(+) to place a 6·His tag at the NH2terminus of the polypeptide According to the CHLOROP prediction [30] the putative mature protein started at position 36 from the first methionine [19] The 5¢-oligonucleotide P1 (5¢-CACTAC TCACAATGCTACTCCATTTCTCTCCG-3¢ and the 3¢-oligonucleotide P2 (5¢-GCATAATCCAGGATCCC TTGTGATCTTAAGATG-3¢) were used for PCR ampli-fication of the full length tyrAAT2 cDNA This 3¢-oligonu-cleotide is complementary to the beginning of the 3¢UTR, and introduced a BamHI restriction site (underlined) The full-length PCR fragment was cloned into the plasmid pPCR-Script (Stratagene) Constructs lacking the transit peptide sequence were produced according to theCHLOROP prediction [30] by replacing the arginine in position 36 by a methionine We used P3 (5¢-CTCTTCGAATTCATATG ATCGACGCCGCCC-3¢) that introduced an ATG initi-ator and a NdeI restriction site (underlined) in position 108 fromthe first in frame ATG codon and P2 Then the resulting PCR NdeI–BamHI DNA fragment was cloned into the plasmid pET21 a(+), yielding the plasmid pET21-TyrAAT2
For both constructs, sequencing of the entire insert was carried out and was in complete agreement with the expected sequences The theoretical masses of the products TyrAAT1 and TyrAAT2 were determined to be 68.5 kDa and 36 kDa, respectively
Expression and purification of recombinant TyrAAT1 The E coli AT 2471(kDE3) cells transformed with pET28-TyrAAT1were grown at 37C in Luria–Bertani medium supplemented with the appropriate antibiotics When D600 reached 0.6, 1 mM of isopropyl thio-b-D-galactoside was added to induce recombinant protein synthesis The cells were further grown for 16 h at 28C, harvested, and centrifuged for 20 min at 4000 g The pellet was resus-pended in buffer A (20 mM Tris/HCl, pH 7.9, 500 mM NaCl) containing 5 mM imidazole and sonicated with a Vibra-cell disrupter (Sonics and Materials, Danbury, CT, USA) (100 pulses every 3 s on power setting 5) The crude extract obtained was centrifuged for 20 min at 18 000 g The soluble protein extract was applied to a Ni-nitrilo-triacetic acid agarose column (1.6· 3 cm; Qiagen) column previously equilibrated with 30 mL of buffer A containing
5 mM imidazole The column was washed with buffer A containing 60 mMimidazole The recombinant protein was eluted using buffer A containing 250 mMimidazole Frac-tions containing the major arogenate dehydrogenase acti-vity were pooled and dialyzed against buffer A Enzyme was stored at 4C without loss of activity during several months
Fig 1 Biosynthesis pathway leading to tyrosine and phenylalanine The
alternative for the synthesis of tyrosine and phenylalanine
frompre-phenate are represented EPSP synthase, 5-enolpyruvyl shikimate
3-phosphate synthase.
Trang 3Expression and purification of recombinant TyrAAT2
The E coli AT 2471(DE3) strain transformed with
pET21-TyrAAT2were grown at 37C in Luria–Bertani medium
supplemented with the appropriate antibiotics When D600
reached 0.6, 1 mM of isopropyl thio-b-D-galactoside was
added to induce recombinant protein synthesis The cells
were further grown for 16 h at 28C, harvested, and
centrifuged for 20 min at 4000 g The pellet was resuspended
in buffer B containing 50 mM Tris/HCl, pH 7.5, 1 mM
EDTA, 1 mM dithiothreitol, 1 mM benzamidine HCl,
5 mMamino caproic acid, and sonicated with a Vibra-cell
disrupter (Sonics and Materials) (100 pulses every 3 s on
power setting 5) The crude extract obtained was centrifuged
for 20 min at 18 000 g
The soluble protein extract was subjected to
ammo-niumsulfate fractionation by addition of solid (NH4)2SO4
(20% saturation) at 4C After 20 min of stirring, the
mixture was centrifuged at 40 000 g for 20 min and the
supernatant was brought to 40% saturation with solid
(NH4)2SO4 at 4C The precipitate was recovered by
centrifugation and resuspended in a minimum volume of
buffer A The resulting protein extract was applied to a
Hiload Superdex S200 (3.2· 60 cm, Pharmacia), column
connected to an FPLC system(Pharmacia) previously
equilibrated in 200 mL of buffer B Fractions enriched
with arogenate dehydrogenase were pooled and the
enzyme was stored at 4C without loss of activity during
several month
Protein concentration
Total protein concentration was determined with the
Bio-Rad protein assay with c-globulin as the standard, as
described by Bradford [25] The concentration of purified
arogenate dehydrogenase was also determined by
measur-ing the absorbance at 205 nm[26]
Electrophoretic analyses of proteins
For the analysis of the protein purification, polypeptides
were separated by SDS/PAGE containing 12% (w/v)
acrylamide as detailed by Chua [27] and visualized by
staining with Coomassie Brilliant Blue R250
Enzyme activities
To monitor protein purification, prephenate and
arogen-ate dehydrogenase activities were assayed according to
Bonner & Jensen [29] by following at 25C the form ation
of NADH or NADPH at 340 nmin a buffer containing
50 mM Tris/HCl, pH 7.5, 300 lM prephenate or
arogen-ate, and 1 mM NAD or NADP in a total volume of
200 lL
Enzyme activity is expressed in UÆmg)1, were 1 U is
defined as 1 lmol NAD(P)H formed per min
Data analysis
Kinetic data were analyzed withKALEIDAGRAPHprogram
(Abelbeck Software) providing an iterative fit to the
appropriate equation by using a nonlinear curve-fitting
method
Hyperbolic curve were fitted to the following equation:
v¼ V
app
m :½S
And sigmoidal curve were fitted to the following equation:
v¼ V
app
m :½Sn
where Vapp
m and Kapp
m are the apparent Vmand Kmvalues for one substrate at different fixed value for the concentration
of the second substrate and n is the Hill coefficient
R E S U L T S Two forms of arogenate dehydrogenase were previously identified in the A thaliana databanks, tyrAAT1 (accession number AF434681), and tyrAAT2 (accession number AF434682) [19] tyrAAT1 was previously described under the name tyrAATc [19] According to theCHLOROP predic-tion program[30], the two forms present a putative plastidic transit peptide sequence This finding is in good agreement with a plastidic localization of all other enzyme activities involved in aromatic amino acids biosynthesis (reviewed in [16,17]) We have reported previously that TyrAAT1 is comprised of two highly similar peptide domains Tyr-AAT1.1 and TyrAAT1.2 (Fig 2A) A biochemical analy-sis carried out on crude protein extracts revealed that both domains possess arogenate dehydrogenase activity [19] The second isoform, TyrAAT2, does not contain repeated domains (Fig 2A) Sequence comparison between TyrAAT2 and each of the two peptide domains of TyrAAT1 revealed more than 50% identity in the two cases (Fig 2B)
Purification of TyrAAT1 and TyrAAT2 The two recombinant isoforms of A thaliana arogenate dehydrogenase TyrAAT1 and TyrAAT2 lacking their putative plastidic transit peptide were overproduced in the
E coli mutant strain AT 2471(DE3) cells devoid of endogenous prephenate dehydrogenase activity [23] Both isoforms were purified to near homogeneity (more than 95% of purity) as assessed by denaturing PAGE and visualization by Coomassie Blue staining As documented in Fig 3, in denaturing condition both purified proteins have the expected molecular mass deduced from their coding sequences, i.e 66 vs 68 kDa and 38 vs 37 kDa for TyrAAT1 and TyrAAT2, respectively Recombinant TyrAAT1 was never recovered in large amounts in the soluble protein fraction [19], we thus decided to carry out a purification of a recombinant His-tagged TyrAAT1 in a one-step procedure via Ni-nitrilotriacetic acid affinity chro-matography This purification process resulted in a 700-fold enhancement of the specific activity (Table 1) About 1.4 mg of enzyme was routinely obtained from 1 g of soluble proteins TyrAAT2 was expressed without its transit peptide as described in Materials and methods The resulting protein was successfully overproduced in AT 2471(DE3) cells and was recovered mainly as soluble protein Recombinant TyrAAT2 arogenate dehydrogenase was excluded froma gel filtration S200 column (Fig 3B) Therefore, a two step purification strategy was adopted for
Trang 4this enzyme and consisted of precipitation in a 20–40%
ammonium sulfate followed by chromatography on a S200
gel filtration column (Fig 3A, Table 1) This purification
resulted in a 12-fold enhancement in specific activity About
17 mg of enzyme was routinely obtained from 1 g of soluble
protein by this procedure
Size exclusion chromatography using Superdex S200
revealed that TyrAAT1 eluted with a mobility consistent
with that of a monomeric protein of 67 kDa (Fig 3B), and
in total agreement with the mass estimated by denaturing
PAGE (i.e 66 kDa, Fig 3A) In contrast, TyrAAT2 eluted
in the void volume of the S200 column even in presence of
300 mMNaCl (Fig 3B) A similar behavior was found with
the TyrAAT2 arogenate activity found in crude protein
extract of cultured cells from A thaliana This finding,
Fig 2 Schematic diagram of the amino acid sequence of the two
A thaliana arogenate dehydrogenases TyrAAT1 and TyrAAT2 deduced
from their respective coding sequences (A) and alignment of amino acid
sequence of the two protein domains of TyrAAT1 (TyrAAT1.1, and
TyrAAT1.2), and TyrAAT2 using the CLUSTALW program (B) (A)
Putative transit peptide cleavage site determined using CHLOROP
pro-gramare indicated (i.e Val36 for TyrAAT1 and Arg 36 for TyrAAT2).
(B) Conserved residues are noted by *, and high similarity residues are
noted by colons.
Fig 3 Purification for the two A thaliana arogenate dehydrogenases TyrAAT1 (left) and TyrAAT2 (right) monitored by SDS/PAGE (A) and behavior of TyrAAT1 and TyrAAT2 by chromatography on Superdex 16/60 S200 (B) (A) Polypeptides were separated on SDS/ PAGE gels and stained with Coomassie brillant blue R-250 Lane 1: crude soluble extract (15 lg) of E coli AT 2471(DE3) cells trans-formed with pET28-TyrAAT1 Lane 2: Fraction containing TyrAAT1 eluted froma Ni-nitrilotriacetic acid agarose column by a 250-m M imidazole buffer (1 lg) Lane 3: crude soluble extract (15 lg) of E coli AT 2471(DE3) cells overproducing TyrAAT2 Lane 4: ammonium sulfate 20–40% fractionation of crude soluble extract of E coli AT 2471(DE3) cells overproducing TyrAAT2 (10 lg) Lane 5: Fraction containing TyrAAT2 eluted froma S200 gel filtration column (2 lg) The minor polypeptide of 72 kDa resolved in lane 5 correspond to a dimeric form of TyrAAT2 This was determined by Western blot analysis MW: molecular mass markers (B) A 0.5-mL sample of purified TyrAAT1 or TyrAAT2 (1 mg) were applied to the S200 column and eluted at 1.5 mLÆmin)1
in a buffer containing 50 m M Tris/HCl, pH 7.5 The straight line represents the profile of activity obtained with TyrAAT1 and the dashed line the profile of activity obtained with TyrAAT2 TyrAAT2 eluted in the void volume of the S200 TyrAAT1 eluted with a Ve/Vo of about 1.7 which correspond to an apparent mass
of 67 kDa as determined by comparison with mobility of standard protein (insert panel) Standards used (1 mg) were thyroglobulin (669 kDa), apoferritin (443 kDa), b-amylase (200 kDa), alcohol dehydrogenase (150 kDa) and bovine serumalbumin (66 kDa).
Trang 5together with the result fromSDS/PAGE, indicates that
native and recombinant TyrAAT2 form oligomers with a
molecular mass greater than 600 kDa
Kinetic analyses
The two enzymes are strictly NADP-dependent Both forms
obeyed Michaelis–Menten behavior at saturating
concen-trations of the second substrate The presence of a His-tag at
the NH2terminus of TyrAAT1 did not modify its kinetics
properties as its Kmvalues for NADP and arogenate were
very similar to the values previously determined with the
unpurified recombinant protein devoid of His-tag [19]
Michaelis–Menten constants for arogenate and NADP are
presented in Table 2 Kmvalues for NADP were found to be
10.2 lMand 14.3 lMfor TyrAAT1 and TyrAAT2,
respect-ively They are in the same order of magnitude as that
previously reported for unpurified plant arogenate
dehy-drogenases [5,6,19,31] The Kmvalues for arogenate were
52.6 and 84.2 lMfor TyrAAT1 and TyrAAT2, respectively
(Table 2) The Km values for arogenate reported in the
literature for other plant enzymes differ significantly They
range from67 to 340 lM[4–6,19,31]
Values for the maximal velocity (Vm) obtained for
purified TyrAAT1 and TyrAAT2 were 142 and 73 UÆmg)1,
respectively They are higher than those reported for
prephenate dehydrogenase [3,21], and are of the same order
of magnitude as that reported for the arogenate
dehydrog-enase purified from Phenylobacterium immobile [12], and
from Synechocystis (Rippert & Matringe, in preparation)
The Vm of TyrAAT1 is twice that of TyrAAT2 If we
consider that the two active sites of TyrAAT1 are
equivalent, values for the turn over number (kcat) were determined as 84 s)1 for one domain of TyrAAT1 and
37 s)1for one TyrAAT2 monomer Values for the catalytic efficiency (kcat/Km) for arogenate were estimated at 1.6· 106M)1Æs)1 and 0.44· 106M)1Æs)1 for TyrAAT1 and TyrAAT2, respectively (Table 2) The two isoforms differ also with respect to their capacity to use prephenate as
an alternative substrate TyrAAT1 was incapable of cata-lyzing the transformation of prephenate to p-hydroxyphe-nylpyruvate regardless of the concentration of prephenate tested (i.e up to 1 mM) However, prephenate at high concentrations could bind to the active site as it behaves as a competitive inhibitor with respect to arogenate (not shown) Its apparent Kivalue was estimated at 4.2 mM(Table 3) In contrast, the second isoformTyrAAT2 exhibits weak prephenate dehydrogenase activity Fromour data, its Km and kcatvalues could be estimated as 17 mM and 3.4 s)1, respectively, and the catalytic efficiency for prephenate (kcat/Km) was 2· 102M )1Æs)1 (Table 2) Therefore, at nonsaturating concentration of prephenate (< 17 mM), the reaction is 2000 times less efficient in catalyzing the reaction with prephenate than with arogenate (0.44· 106
M )1Æs)1 vs 2· 102
M )1Æs)1) (Table 2) This prephenate dehydrogenase activity could not be attributed
to an endogenous E coli activity that would coelute with arogenate dehydrogenase as the recombinant TyrAAT2 protein was overproduced in the E coli strain AT 2471, devoid of prephenate dehydrogenase activity [23] As expected for an alternate substrate, prephenate at high concentrations exhibited competitive inhibition with respect
to arogenate (not shown) An apparent Ki value for prephenate was determined to 2.4 mM(Table 3) Finally,
Table 2 Kinetic properties of the two purified A thaliana arogenate dehydrogenases TyrAAT1 and TyrAAT2 KALEIDAGRAPH programwas used to analyze data Hyperbolic curves were fitted to Eqn (1) To determine K m NADP, plots of the velocity as a function of increasing concentrations of NADP (8–1000 l M ) at a fixed concentration of arogenate (300 l M ) was used To determine K m arogenate, plots of the velocity as a function of increasing concentrations of arogenate (0–300 l M ) at a fixed concentration of NADP (1000 l M ) were used K m values were determined using Eqn (1) with a nonlinear curve-fitting of plots.
k cat /K m arogenate ( M )1 Æs)1) (1.6 ± 0.3) · 10 6
(4.4 ± 0.4) · 10 5
Table 1 Purification of the two A thaliana arogenate dehydrogenases TyrAAT1 and TyrAAT2 NTA, nitrilotriacetic acid.
Total protein (mg)
Total activity (U)
Specific activity (UÆmg protein)1)
Recovery (%)
Purification -fold TyrAAT1
TyrAAT2
Trang 6the arogenate activities of both isoforms were totally
insensitive towards concentration up to 1 mMof
p-hydroxy-phenylpyruvate, phenylalanine or tryptophan, and highly
sensitive towards inhibition by tyrosine, the product of the
reaction (see below)
Kinetic mechanism
Steady-state velocity studies in the absence of product
The kinetic mechanism of both enzymes was investigated
by varying the concentration of one substrate at fixed
concentrations of the second substrate For both enzymes,
initial velocity patterns obtained by varying the
concentra-tions of NADP (4–500 lM) at fixed concentrations of
arogenate (90–250 lM) were linear and intersected to the left
of the vertical axis (Fig 4B,D) Similar patterns were
obtained by varying the concentrations of arogenate
(10–250 lM) at fixed concentrations of NADP
(50–100 lM) (data not shown) Thus, under these
condi-tions, the reaction conforms to a simple sequential
mech-anismfor both isoforms However, when NADP is used at
low concentrations (between 10 and 30 lM), with arogenate
as the variable substrate sigmoı¨dal, curves were obtained
(Fig 4A,C), indicating positive kinetic cooperativity with
respect to the binding of arogenate for both enzymes When
the data in Fig 4A and C were fitted to the Hill equation,
Hill coefficient values of 1.8 and 1.6 were obtained for
TyrAAT1 and TyrAAT2, respectively When NADP was
the variable substrate, in both cases, double reciprocal plots were linear (Fig 4B,D) Thus there is no kinetic coopera-tivity for the interaction of NADP with the enzyme
Table 3 Values for the apparent inhibition constant of the two arogenate dehydrogenases TyrAAT1 and TyrAAT2 by product and substrate analogues The concentrations of each inhibitor and fixed or varied substrate used are given in the table ND, not determined; C, competitive; NC, noncompetitive; M, mixed type inhibition Values for the apparent inhibition were obtained using equations (a), (b) and (c) for competitive, noncompetitive and mixed inhibition, respectively K m and V m values used in equation were directly determined from hyperbolic curve or sigmoidal curve using Eqns (1) and (2).
(a) K i ¼ Vmax:½I
V m V mapp
(b) K i ¼ Km:½I
K mapp K m
and (c) K i ¼ ½I
Slope 1
½s V max
Kmax V app max
Inhibitor
Substrate
Type of inhibition
NADPH (0, 50, 100 l M ) Arogenate NADP (20 l M ) NC TyrAAT1 53.9 ± 11
TyrAAT2 ND NADP Arogenate (200 l M ) C TyrAAT1 58.8 ± 6
TyrAAT2 ND Tyrosine (0, 50, 100 l M ) Arogenate NADP (50 l M ) C TyrAAT1 8.1 ± 0.3
TyrAAT2 7.5 ± 0.4 NADP Arogenate (70 l M ) NC TyrAAT1 14.2 ± 0.4
TyrAAT2 16.6 ± 3.4 AMP (0, 10, 20 l M ) Arogenate NADP (50 l M ) M TyrAAT1 25 900 ± 900
TyrAAT2 17 800 ± 6200 NADP Arogenate (200 l M ) C TyrAAT1 115 000 ± 9000
TyrAAT2 73 900 ± 11 000 Prephenate (0, 1, 2 m M ) Arogenate NADP (100 l M ) C TyrAAT1 4200 ± 800
TyrAAT2 2400 ± 400 NADP Arogenate (70 l M ) M TyrAAT1 20 200 ± 1100
TyrAAT2 22 500 ± 2500 cis-Aconitate (0, 10, 20 l M ) Arogenate NADP (100 l M ) C TyrAAT1 27 700 ± 5100
TyrAAT2 5800 ± 800 NADP Arogenate (200 l M ) M TyrAAT1 42 900 ± 9600
TyrAAT2 25 300 ± 2000
Fig 4 Effects of substrate concentration on the velocity of the two arogenate dehydrogenases TyrAAT1 (upper panels), and TyrAAT2 (lower panels) (A) and (C) Plots of the velocity as a function of increasing concentrations of arogenate (0–250 l M ) at fixed concen-trations of NADP (10, 20 and 30 l M ) Sigmoidal curves were fitted to Eqn (2) using KALEIDAGRAPH program (B) and (D) Lineweaver–Burk representations of velocity as a function of increasing concentrations of NADP (8–1000 l M ) at fixed concentrations of arogenate (20, 30 and
40 l M ).
Trang 7Steady-state velocity studies in the presence of product
andproduct or substrate analogues Patterns of inhibition
by products and substrate or product analogues were
constructed to determine if the reactions catalyzed by
TyrAAT1 and TyrAAT2 conformto a rapid equilibrium
randomor steady-state ordered kinetic mechanism
Inhibi-tion by NADPH was monitored in the presence of 20 lM
NADP as no inhibition was observed at saturating
concentrations of NADP At 20 lM of NADP double
reciprocal plots were concave up due to the positive
cooperativity for the interaction of arogenate with
TyrAAT1 Replots of 1/v vs 1/s2 were used to linearize
the curves (Fig 5Ai) [32] Inhibition by NADPH was linear
noncompetitive with respect to arogenate, and competitive
with respect to NADP (Fig 5Ai,ii) Kivalues for NADPH
were found to be about 58 lM (Table 3) For TyrAAT2,
nonlinear plots were also observed when varying arogenate
at a fixed concentration of NADP However, NADPH did
not inhibit the reaction at the concentrations tested (not
shown), indicating that TyrAAT2 was less sensitive towards
inhibition by NADPH than TyrAAT1
Lineweaver–Burk plots revealed that for both enzymes, tyrosine causes linear competitive inhibition with respect to arogenate (Fig 5Bi,iii), and noncompetitive inhibition with respect to NADP (Fig 5Bii,Biv) Kivalues for tyrosine were found to be about 8 and 7 lMfor TyrAAT1 and TyrAAT2, respectively (Table 3)
In order to confirm the kinetic mechanism, the inhi-bitory effect of AMP and cis-aconitate as potential analogues of NADP and arogenate, respectively [21] were also tested Lineweaver–Burk plots revealed that AMP acts as a dead-end inhibitor, giving rise to inhibition that is linear competitive with respect to NADP, and linear mixed-type inhibition with respect to arogenate cis-Aconi-tate exerts a competitive inhibition with respect to arogenate and mixed-type competitive with respect to NADP (not shown) As reported above, for both isoforms prephenate acts as a competitive inhibitor, with respect to arogenate, and noncompetitive with respect to NADP (not shown)
D I S C U S S I O N The shikimate pathway plays a pivotal role in providing the cell with three aromatic amino acids tyrosine, phenyl-alanine and tryptophan, and most of the aromatic secondary metabolites [18] In certain circumstances, for example in woody plants, at least 30% of the carbon fixed during photosynthesis is incorporated by this pathway for the synthesis of lignin via phenylalanine [33] Detailed kinetics analysis of branch point enzymes of this pathway
is thus absolutely required for understanding the partition
of the carbon flux between the different end products The present study is the first to report the purification and detailed kinetic characterization of A thaliana arogenate dehydrogenase Both isoforms exhibit initial velocity patterns in the absence and presence of products and dead end inhibitors consistent with a rapid equilib-riumrandomkinetic mechanismand the formation of two dead-end complexes, enzyme–NADP–tyrosine and enzyme–NADPH–arogenate Conclusions about the order
of product release are limited because it was not possible
to determine the inhibition patterns with bicarbonate (CO2)
A similar kinetic mechanism has also been reported for the NAD-dependent prephenate dehydrogenase activity of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase from Aerobacter aerogenes and E coli [3,21] The two monofunctional plant enzymes also exhibit positive kinetic cooperativity in the binding of arogenate, paralleling those findings for the interaction of prephenate with chorismate mutase-prephenate dehydrogenase [35] NADP increases the apparent affinity of the plant enzymes for arogenate but arogenate did not alter the affinity of the enzymes for NADP Both A thaliana arogenate dehydro-genases were very sensitive toward inhibition by the product
of the reaction (tyrosine); in fact they exhibit a higher affinity for tyrosine, than for their arogenate substrate This strong feedback inhibition arises fromthe necessity of the plant cell, to regulate the flux of arogenate between the synthesis of tyrosine and that of phenylalanine Indeed, the monofunctional plants arogenate dehydrogenase and
E coli chorismate mutase-prephenate dehydrogenase are
at the branch point of the synthesis of tyrosine and
Fig 5 Product inhibition of the two arogenate dehydrogenases
Tyr-AAT1 and TyrAAT2 (A) (i) plot representing 1/v function of
1/[aro-genate]2(4–400 l M ) at fixed concentrations of NADP (20 l M ) and at
fixed concentrations of NADPH (0, 50 and 100 l M ) (A) (ii)
Line-weaver–Burk representation of velocity as a function of increasing
concentrations of NADP (3–1000 l M ) at fixed concentrations of
aro-genate (200 l M ) and at fixed concentrations of NADPH (0, 50 and
100 l M ) (B) (i) and (iii) Lineweaver–Burk representations of velocity
as a function of increasing concentrations of arogenate (5–400 l M ) at
fixed concentrations of NADP (100 l M ) and at fixed concentrations of
tyrosine (0, 50 and 100 l M ) B (ii) and (iv) Lineweaver–Burk
repre-sentations of velocity as a function of increasing concentrations of
NADP (5–1000 l M ) at fixed concentrations of arogenate (70 l M ) and
at fixed concentrations of tyrosine (0, 5 and 10 l ).
Trang 8phenylalanine and they are both inhibited by tyrosine
[5,6,36] However, in the case of E coli enzyme tyrosine
binds to an allosteric site and an active tyrosine–enzyme–
prephenate complex could be formed [37] Under these
conditions, complete inhibition by tyrosine would never be
reached even at high tyrosine concentrations In contrast,
the plant enzymes could be completely inhibited by tyrosine
as tyrosine is a competitive inhibitor with respect to
arogenate When it is required, this allows the plant to
utilize the majority of the arogenate formed for the synthesis
of phenylalanine In Synechocystis and Actinomyces
mis-souriensis, arogenate dehydrogenase is not at branch point
between tyrosine and phenylalanine as phenylalanine is
synthesized via phenylpyruvate Accordingly, these enzyme
activities are not regulated by tyrosine [10,38] (P Rippert &
M Matringe, unpublished results)
The comparison of the kinetics properties of these two
isoforms revealed that the peculiar structure of TyrAAT1,
i.e its repeated peptide domains, confers to this enzyme a
catalytic efficiency (kcat/Km) four times greater than that of
TyrAAT2 (Table 2) This may have important
physiologi-cal significance in the partition of the flux of arogenate into
tyrosine or phenylalanine because arogenate
dehydrogen-ases are branch point enzymes in competition with
arogen-ate dehydratases for arogenarogen-ate (Fig 1) The lower substrarogen-ate
specificity of TyrAAT2, probably explains its lower
effi-ciency in catalyzing the arogenate dehydrogenase reaction
The high Kmfor prephenate and the low specific activity of
this prephenate activity, indicate that it is a side reaction
with no physiological significance Interestingly, a BLAST
search allowed us to identify a gene fragment from Brassica
oleracea(accession number BH495674), encoding a putative
arogenate dehydrogenase with two highly similar peptide
domains, indicating that this peculiar structure is not
restricted to A thaliana, and may also be present in other
plant species Immunological analyses, which are presently
under investigation in our laboratory, will help us to address
this issue
The presence of two plastidic isoforms with different
kinetic behavior raised the question of their respective
physiological roles The presence of several plastidic
isoforms is a general feature for the enzymes of the
aromatic amino acid pathway Indeed, the complete
sequence of the A thaliana genome [20] revealed two
plastidic isoforms for 5-enolpyruvyl shikimate 3-phosphate
synthase and chorismate mutase, three for
2-ceto-3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and
shiki-mate kinase, and not less than six for arogenate
dehydratase Analysis of the patterns of expression of
the two A thaliana arogenate dehydrogenases in different
organs and in response to different environmental
condi-tions by real-time PCR will help us to address the
question of their physiological roles These studies are
underway in our laboratory
A C K N O W L E D G E M E N T S
We are grateful to Roland Douce, Claude Alban, Gilles Curien and
Renaud Dumas for critical reading of the manuscript This work was
supported by Centre National de la Recherche Scientifique, by the
Institut National de la Recherche Agronomique and by Aventis
CropScience.
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