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INTRODUCTION |
Chorismate synthase catalyzes the final step in the shikimate
pathway, which links the metabolism of carbohydrates to the biosynthesis of the three aromatic amino acids and many aromatic secondary metabolites in a series of seven enzymatic steps. The pathway
is absent from mammals making it a prime target for the development of
antimicrobial and herbicidal agents. Chorismate synthase itself is
biochemically unique in nature in that it catalyzes a
1,4-anti-elimination of the 3-phosphate group and the 6 (pro-R)-hydrogen from 5-enolpyruvylshikimate 3-phosphate
(EPSP)1 to yield chorismate
(1, 2). There is no other example of this type of catalysis known in
nature, thereby making it exclusive. The enzyme has an absolute
requirement for reduced FMN (3, 4) and can be classified with regard to
how it acquires this essential cofactor. The chorismate synthases for
which the reduced flavin has to be supplied exogenously are referred to
as monofunctional, e.g. those from Escherichia
coli and plants (3, 5-9), while chorismate synthases which
possess the intrinsic ability to reduce the flavin at the expense of
NADPH are referred to as being bifunctional, e.g. the
Neurospora crassa enzyme (4, 10, 11). From an evolutionary
point of view, while it has been concluded from a phylogenetic analysis
that all chorismate synthases are of monophyletic origin, it is not
clear if the ancestral chorismate synthase was mono- or bifunctional
(12). It has been suggested that the ancestral enzyme harbored the
intrinsic flavin reductase activity (i.e. was bifunctional)
as it is hard to imagine how this activity could have evolved in a
framework of what are known to be monofunctional enzymes,
i.e. the bacterial and plant chorismate synthases (12). In
any case, as reduction of the FMN cofactor could be envisioned as a
possible regulatory effector of chorismate synthase it would be
intriguing to establish the history of how the enzyme has maintained reduction of the cofactor.
Recently, the genome of the hyperthermophilic bacterium
Thermotoga maritima (Tmax = 90 °C,
Topt = 80 °C) has been completed and 24% of
its genes have been reported to be more similar to archaeal genes than
to other bacterial genes (13). As this is a much higher percentage than
that found in mesophilic bacteria (3-7%), it has been suggested that
considerable lateral gene transfer has occurred between archaea and
T. maritima (13). In fact, T. maritima is
believed to be one of the oldest and most slowly evolving lineages in
the eubacteria (14). Therefore, classification of T. maritima chorismate synthase with regard to how it acquires the
reduced FMN cofactor can be considered to reflect the nature of the
ancestral enzyme. In addition, thermophilic proteins, as a result of
residing in an extreme environment, are more stable and tend to display
greater conformational rigidity than mesophilic proteins. Therefore, a
comparison of the characteristics of a thermophilic chorismate synthase
with that of a mesophilic counterpart may provide insights not only
into the stability but also on the self-organization of this enzyme.
Here we report the cloning, expression, and purification of chorismate
synthase from the thermophilic eubacterium T. maritima. This
is the first time that this enzyme has been described from an
extremophilic organism. We show that the enzyme is monofunctional with
respect to its requirement for FMN indicating that the ancestral
protein was in fact monofunctional rather than bifunctional as had been
proposed previously (12). In addition, we describe the characteristics
of the thermophilic enzyme compared with those of the mesophilic
E. coli chorismate synthase. In particular, we address the
thermal stability as well as the spectrophotometric and electrophoretic
properties of both the mesophilic and thermophilic enzymes in the
presence and absence of ligands. We also report on the quaternary
structure of T. maritima chorismate synthase.
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EXPERIMENTAL PROCEDURES |
Chemicals--
Restriction endonucleases and DNA modification
enzymes were purchased from Roche Molecular Biochemicals (Mannheim,
Germany) or New England Biolabs (Beverly, MA). Oligonucleotide
sequencing and polymerase chain reaction primers were obtained from
Microsynth (Balgach, Switzerland). All other chemicals were of the
highest grade available.
Molecular Techniques--
Basic molecular techniques were
adopted from Ausubel et al. (15) or Sambrook et
al. (16).
Cloning and Expression of T. maritima Chorismate
Synthase--
Cell paste (1 g) of the thermophilic organism T. maritima (strain MSB8) was kindly provided by Dr. Huber
(University of Regensburg, Germany). The genomic DNA was isolated using
the QIAamp Tissue kit (Qiagen) according to the instructions described
by the manufacturer. The DNA sequence of the T. maritima
aroC gene which codes for chorismate synthase was obtained from
GenBankTM (accession number AE001715.1). The
aroC gene was amplified by the polymerase chain reaction
(PerkinElmer Life Sciences) (5'-primer, GGAATTCCATATGAAACTCACGATAGCGGGGGATTCC; 3'-primer,
CCCAAGCTTCTACCGATAGTGCTCTTTCCAGAACCC) based on the MSB8 strain sequence
and cloned into the NdeI and HindIII restriction
sites, respectively, of pET21a (Novagen). This vector allows expression
of T. maritima chorismate synthase under control of the IPTG
inducible T7 promoter. The expression construct was verified by
sequence analysis and transformed into either E. coli
BL21(DE3) or E. coli BL21-CodonPlusTM (DE3)-RIL
cells (Stratagene). For analysis of expression, a single transformant
was grown in 5 ml of 2 × YT medium supplemented with 100 mg/liter
ampicillin and this was used to inoculate a 50-ml culture. After 1 h at 37 °C, expression was induced by addition of IPTG to a final
concentration of 0.1 mM. Cells were allowed to grow for
another 5 h at 37 °C and were then harvested by centrifugation and subsequently stored at
80 °C.
Protein Purification--
E. coli cells (80 g) were
resuspended in 20 ml of Buffer A (50 mM Tris-HCl, pH 7.5, containing 50 mM potassium chloride, 10% glycerol, 0.4 mM dithiothreitol, 1.3 mM EDTA, and 1.3 mM phenylmethylsulfonyl fluoride). Lysozyme was added to a
final concentration of 1 mg/ml and after 20 min at room temperature the
cells were further lysed by sonication. The cell debris was removed by
centrifugation at 25,000 × g for 30 min at room
temperature. The supernatant was heated at 75 °C for 15 min after
which the precipitated protein was removed by centrifugation
(25,000 × g for 15 min at room temperature). The
supernatant from the heat treatment was then subjected to anion
exchange chromatography on a DEAE-Sephacel column (2.5 × 16 cm)
freshly equilibrated in Buffer A. After washing with Buffer A, protein
was eluted by a linear gradient of 250 ml of Buffer A and 250 ml of
Buffer B (50 mM Tris-HCl, pH 7.5, containing 200 mM potassium chloride, 10% glycerol, 0.4 mM
dithiothreitol, 1.3 mM EDTA, and 1.3 mM
phenylmethylsulfonyl fluoride). The progress of protein purification
was monitored by 10% SDS-PAGE using the buffer system as described by
Laemmli (17).
N-terminal Amino Acid Sequencing--
Either a crude extract (as
outlined above) from IPTG induced expression of E. coli BL21
CodonPlus(DE3)-RIL cells harboring the constructed pET-T.
maritima aroC plasmid or the purified protein was subjected to
10% SDS-PAGE. The electrophoresed sample was blotted onto a
polyvinylidene difluoride membrane (Bio-Rad) according to the
manufacturer's recommendations. The band of interest was excised and
subjected to automated Edman degradation in an Applied Biosystems 477A sequencer.
Immunochemical Methods--
For Western blot analysis, the
samples were separated by 10% SDS-PAGE and blotted onto a
nitrocellulose membrane. The membrane was blocked with TBS (10 mM Tris-HCL, pH 7.5, 0.9% sodium chloride) supplemented
with 5% dry skimmed milk and 0.05% Tween 20. The blot was incubated
for 90 min with an affinity purified antibody raised against chorismate
synthase from the higher plant Corydalis sempervirens at a
suitable dilution in the blocking buffer. The membrane was washed five
times for 10 min each in TBST (TBS containing 0.05% Tween 20). It was
then incubated for 45 min in TBST containing 5% dry skimmed milk and a
sheep anti-rabbit peroxidase conjugate (Roche Molecular Biochemicals)
diluted 1:3000. After another five washes of 10 min each in TBST, the
blot was developed using the chemiluminescent system and according to
the protocol supplied by Roche Molecular Biochemicals.
Enzyme Assays--
Chorismate synthase activity was assayed
using forward coupling of the reaction to anthranilate synthase at
30 °C essentially as described by Schaller et al. (7),
except that the assay mixture used was 0.1 M potassium
phosphate, pH 7.6, containing 30 mM ammonium sulfate, 10 mM glutamine, 4 mM magnesium sulfate, 1 mM dithiothreitol, 10 µM FMN, 50 picokatal
anthranilate synthase (from E. coli), and 80 µM EPSP. Either 5 mM sodium dithionite or 1 mM NADPH were used to start the reaction.
CD Spectroscopy--
CD measurements were performed with a
Jasco-715 spectropolarimeter equipped with a computer controlled water
bath, using thermostatted cuvettes of 0.2-mm path length. Thermal
unfolding curves were measured by continuously measuring the
ellipticity at 222 nm between 3 and 92 °C at a scan rate of 1.0 degree min
1 and with data collection every 20 s.
Protein samples were prepared for CD spectroscopy in 10 mM
HEPES, pH 7.4, containing 50 mM ammonium sulfate.
PAGE--
Native PAGE was performed using the Pharmacia
PhastSystem with an 8-25% gel run at pH 8.8 for 240 A·V·h
over a period of 45 min at 15 °C. Either oxidized FMN and/or EPSP
were added in 5-fold molar excess to samples of enzyme (13 µM, in 10 mM HEPES, pH 7.4, containing 50 mM ammonium sulfate) as indicated in the legend to Fig. 3
and 20 min before beginning electrophoresis. Gels were stained with
Coomassie Blue. The protein standards used were thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), and
bovine serum albumin (66 and 132 kDa).
Analytical Ultracentrifugation--
Conventional
sedimentation equilibrium measurements were made according to the
method of Liu et al. (18) with a Beckman XL-A analytical
ultracentrifuge (Beckman, Palo Alto, CA). The data were collected at
7,000, 9,000, and 12,000 rpm and at 20 °C. The sample volume was 120 µl at a protein concentration of 0.27 mg/ml in 10 mM
Tris, pH 7.4, containing 90 mM potassium chloride. Protein
samples were dialyzed exhaustively in the above buffer before use.
UV-Visible Absorbance Spectrophotometry--
Absorbance spectra
were recorded with a Uvikon 933 spectrophotometer (Kontron Instruments
AG, Zürich, Switzerland) equipped with a Haake D1 waterbath
(Digitana AG, Horgen, Switzerland). The spectra were recorded in 10 mM HEPES, pH 7.4, containing 50 mM ammonium
sulfate and by systematically varying either the substrate concentration or the temperature degree.
Rapid Reaction Spectrophotometry--
Formation and decay of the
flavin intermediate was observed using a stopped flow spectrophotometer
equipped with a thermostatted 1-cm path length cell and a diode array
detector (Spectroscopy Instruments GmbH, D-82205 Gilching)
interfaced with a Macintosh IIcx computer. Data were acquired using
SPECTRALYS 1.55 software (ZINS ZIEGLER-Instruments GmbH, Nobelstrasse
3-5, D-41189 Mönchengladbach). Rapid reactions were recorded
between 300 and 600 nm, the integration time for collecting a spectrum
was 10 ms with a resolution of 2 pixels/nm. FMN was reduced with sodium
dithionite in anaerobic solutions of the substrate and a stoichiometric
concentration of enzyme. The experiments were performed in 10 mM HEPES, pH 7.4, containing 50 mM ammonium sulfate.
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RESULTS |
PCR of the aroC Gene from T. maritima--
Under the conditions
used here, the polymerase chain reaction resulted in the amplification
of a fragment which was ~1150 base pairs in size. Including the
engineered restriction sites (19 base pairs), this fragment thus
corresponds to the predicted size of the encoding aroC gene
from T. maritima (1128 base pairs) based on the DNA sequence
and was confirmed by DNA sequencing analysis.
Expression and Purification of Recombinant T. maritima Chorismate
Synthase--
The PCR product obtained was cloned into the expression
vector pET21a to create the construct pET-TmaroC. Expression
of T. maritima chorismate synthase (41.7 kDa) could be
obtained in E. coli BL21(DE3) cells, as is the conventional
protocol for this system (Fig. 1,
panel A and B, lane 4), but greater expression could be achieved in E. coli
BL21-CodonPlusTM(DE3)-RIL cells (Stratagene) presumably due
to rare codon usage in TmaroC for arginine and isoleucine
residues (Fig. 1, panel A, lane 8). The expression of
T. maritima chorismate synthase in
BL21-CodonPlusTM(DE3)-RIL cells is ~6-fold greater than
that observed in BL21(DE3) (Fig. 1, panel B, compare protein
loaded in lanes 4 and 8). However, expression in
BL21-CodonPlusTM(DE3)-RIL cells appears to be independent
of induction (Fig. 1, panel B, lanes 7 and 8).
The majority of the recombinant fusion protein obtained was insoluble
(Fig. 2, lane 2). The soluble
T. maritima chorismate synthase fraction (Fig. 2, lane
3) was refined in the first instance by a heat precipitation step
which was optimal at 75 °C. At this temperature the majority of the
contaminating proteins precipitated (Fig. 2, lane 4) and
could be removed by centrifugation, while the T. maritima
chorismate synthase remained in solution (Fig. 2, lane 5).
The most prominent contaminating protein remaining at this stage was
chloramphenicol acetyltransferase from E. coli (25.7 kDa),
as determined by N-terminal amino acid sequencing (15 cycles of 50 pmol
of protein resolved the amino acids MEKKITGYTTVDISQ, which are
identical to the data base entry for this protein, accession number
CAA67774), and could be removed by chromatography using DEAE-Sephacel
(Fig. 2, lane 6). The identity of the purified band as
T. maritima chorismate synthase (41.7 kDa) was confirmed by
N-terminal sequencing analysis (15 cycles of 50 pmol of protein
resolved the amino acids MKLTIAGDSHGKYMV, which are identical to the
data base entry for this chorismate synthase, accession number Q9WYI2),
and the protein was estimated to be greater than 95% pure, as judged
from SDS-PAGE (Fig. 2, lane 6). A typical yield of T. maritima chorismate synthase under these conditions was 30 mg from
80 g of E. coli cell paste.

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Fig. 1.
Analysis of the expression of T. maritima chorismate synthase by SDS-PAGE. A,
Coomassie Blue-stained gel; lanes 1-4 are the empty vector
(pET-21a) and the vector containing T. maritima aroC
(pET-TmCS), respectively, u = uninduced and
i = induced (1 mM IPTG) and are as
expressed in E. coli BL21(DE3) cells; lanes 5-8
are as for lanes 1-4 but in E. coli
BL21-CodonPlusTM(DE3)RIL cells. The amount of protein
loaded in each lane is ~6 µg. TmCS, T. maritima chorismate synthase. B, Western blot analysis
of A using an antibody raised against C. sempervirens chorismate synthase. The amount of protein loaded in
lanes 1-4 is ~0.6 µg while in lanes 5-8 it
is ~0.1 µg.
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Fig. 2.
Analysis of the purification of T. maritima chorismate synthase by SDS-PAGE. Lane
1, molecular mass markers as indicated; lane 2, pellet
of crude cell extract after centrifugation; lane 3, supernatant of crude cell extract after centrifugation; lane
4, precipitated protein after heat treatment; lane 5, soluble protein after heat treatment; lane 6, purified
T. maritima chorismate synthase (TmCS) after
DEAE-Sephacel chromatography.
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Requirement of T. maritima Chorismate Synthase for Reduced
FMN--
The purified recombinant T. maritima chorismate
synthase is catalytically active (Table
I) and its specific activity in the presence of dithionite and at 30 °C is calculated as 135.5 nmol·mg
1·min
1. The specific activity
obtained with T. maritima chorismate synthase (Table I) is
lower than that obtained with two mesophilic chorismate synthases
(Table I, N. crassa and E. coli), assayed under
the same conditions, which is at least partly due to nonoptimal
temperature conditions for the thermophilic enzyme. While there was a
2-fold increase in the specific activity of T. maritima
chorismate synthase on performing the reaction at 37 °C (data not
shown), the instability of anthranilate synthase above 40 °C (the
coupling enzyme used in this assay) did not permit determination of the
optimal temperature of T. maritima chorismate synthase using
this assay. In order to determine if T. maritima chorismate
synthase is mono- or bifunctional, the enzyme assay was performed with
either flavin reduced by dithionite exogenously, or adding NADPH to
ascertain if the enzyme has the intrinsic ability to reduce the FMN
cofactor itself. In this way, the relative percentage of the enzyme
activity obtained with the reduced flavin added exogenously to the
intrinsic ability of the enzyme to reduce the FMN cofactor itself is a
measure of its mono/bifunctionality (where approaching 100% indicates
a bifunctional enzyme). As the specific activity obtained for T. maritima chorismate synthase in the presence of NADPH is only
0.5% of that in the presence of dithionite (Table I, T. maritima), it can be concluded that it is monofunctional. The
respective activities of the monofunctional E. coli
chorismate synthase (Table I, E. coli) and the bifunctional N. crassa chorismate synthase (Table I, N. crassa) are indicated as controls.
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Table I
Chorismate synthase (CS) activity in the presence of either dithionite
or NADPH
The specific activities of the chorismate synthases indicated were
determined in the presence of either 5 mM dithionite or 1 mM NADPH at 30 °C in 0.1 M potassium
phosphate, pH 7.6, containing 30 mM ammonium sulfate, 10 mM glutamine, 4 mM magnesium sulfate, 1 mM dithiothreitol, 10 µM FMN, 50 pkat of
anthranilate synthase (from E. coli), and 80 µM EPSP. The results shown are the average of three
determinations under partial anaerobic conditions.
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Determination of the Melting Temperature of Chorismate Synthase in
the Presence and Absence of Ligands--
The stability of both
E. coli and T. maritima chorismate synthase in
the presence and absence of both oxidized FMN and EPSP was examined
using temperature unfolding experiments as measured by CD spectroscopy
(Fig. 3). The melting temperatures of
E. coli chorismate synthase in the absence and presence of
ligands (54.7 ± 0.5 °C and 58.9 ± 0.04 °C,
respectively) indicate a stabilizing effect of the ligands (Fig. 3,
A-C). The melting temperature of T. maritima
chorismate synthase was estimated to be a minimum of 92 °C in both
the presence and absence of ligands (Fig. 3, D-F). However,
the difference (if any) of the melting temperature for this enzyme with
and without ligands could not be determined from these experiments.

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Fig. 3.
Comparison of the thermal unfolding of
E. coli and T. maritima chorismate
synthases in the presence and absence of ligands. A, E. coli
chorismate synthase (5 µM) in the absence of ligands;
B, E. coli chorismate synthase (5 µM) in the presence of oxidized FMN (5 µM)
and EPSP (10 µM); C, overlay of A
and B; D, T. maritima chorismate synthase (5 µM) in the absence of ligands; E, T. maritima chorismate synthase (5 µM) in the presence
of oxidized FMN (5 µM) and EPSP (10 µM);
F, overlay of D and E. All experiments
were carried out in 10 mM HEPES, pH 7.4, containing 50 mM ammonium sulfate. The melting temperatures were
determined from the best fit of the data to the sigmoidal function
f(x)= ((a-d/1 + (x/c)b) + d), where
c = the value at the inflection point.
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Native PAGE of Chorismate Synthase in the Presence and Absence of
Ligands--
Chorismate synthase from E. coli has been
reported to be a homotetramer (156 kDa) and when run on native-PAGE
(8-25%) appears as a diffuse band with a mobility corresponding to an
apparent mass of ~190 kDa (19). When the enzyme is preincubated in
the presence of both oxidized FMN and EPSP and subjected to native-PAGE as for the enzyme alone, there is a marked shift in the mobility toward
the anode (140-150 kDa), which does not occur in the presence of
either substrate alone (19). The same analysis was performed here with
T. maritima chorismate synthase and its properties were compared with those of the E. coli enzyme (Fig.
4). In contrast to the E. coli
enzyme (Fig. 4, E. coli chorismate synthase, lane 1), T. maritima chorismate synthase alone appears as a
sharp band with a mobility corresponding to ~200 kDa (Fig. 4,
T. maritima chorismate synthase, lane 1).
However, when T. maritima chorismate synthase is incubated
in the presence of both oxidized FMN and EPSP, there is a slight shift
in mobility toward the anode akin to what was observed with the
E. coli enzyme but not nearly as pronounced (Fig. 4, compare
lane 4 of E. coli and T. maritima chorismate synthase). Similar to what was observed with the E. coli enzyme, there is no apparent shift in the mobility of
T. maritima chorismate synthase when the enzyme is
preincubated with either of the substrates alone (Fig. 4, compare
lanes 2 and 3 of E. coli and T. maritima chorismate synthase, respectively).

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Fig. 4.
Native-PAGE (8-25%) of chorismate synthase
in the presence and absence of ligands. T. maritima
chorismate synthase: lane 1, T. maritima
chorismate synthase (13 µM); lane 2, T. maritima chorismate synthase (13 µM) and oxidized
FMN (65 µM); lane 3, T. maritima chorismate
synthase (13 µM) and EPSP (65 µM);
lane 4, T. maritima chorismate synthase (13 µM), oxidized FMN (65 µM), and EPSP (65 µM) E. coli chorismate synthase: lanes
1-4 are the same conditions as for the T. maritima
enzyme. S, the standards are as indicated.
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Determination of the Quaternary Structure of Chorismate
Synthase--
The molecular mass of T. maritima chorismate
synthase was estimated by performing sedimentation equilibrium
experiments at a variety of operational speeds in an analytical
ultracentrifuge. A representative data set from a sedimentation
equilibrium experiment is shown in Fig.
5. The data can be interpreted as
resulting from a single ideal species according to the model of Liu
et al. (18) under all the conditions used. The molecular
mass of the enzyme was calculated to be 168,377 ± 1950 Da under
each of the conditions used. The molecular mass of the monomer
estimated from the amino acid sequence is 41,754 Da. In addition, the
enzyme prepared here gave a single homogenous band on SDS-PAGE with a
molecular mass of
42 kDa. Therefore, it can be concluded that
T. maritima chorismate synthase is a tetramer of identical
subunits.

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Fig. 5.
Sedimentation equilibrium analysis of
T. maritima chorismate synthase. The experiment
was performed at 9000 rpm and at 20 °C as described under
"Experimental Procedures." A, data which were
fitted to cr = co·e{Mr,app
(1 2 ) 2(r2 r02)}/2RT, where
c is the concentration (absorbance) at radial position
r, Mr,app the apparent molecular
mass, 2 the partial specific
volume, the solvent density, the angular
velocity and R and T the gas constant and
temperature, respectively. B, computed residuals to the fit
illustrated in A.
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UV-Visible Absorption Spectral Properties of T. maritima Chorismate
Synthase--
Chorismate synthase isolated from T. maritima
under the conditions used here, showed a single absorption maximum at
278 nm (Fig. 6, panel A).
There were no absorbance maxima characteristic for bound flavin (i.e.
370 nm and
450 nm), thus indicating that chorismate synthase is
isolated as the apoenzyme form. Oxidized free FMN (25 µM
in 10 mM HEPES, pH 7.4, containing 50 mM
ammonium sulfate) exhibits absorption maxima at 372 and 445 nm,
respectively (Fig. 6, panel A). Addition of 28 µM chorismate synthase to 25 µM oxidized
FMN at room temperature caused no perturbation of these maxima (Fig. 6,
panel A). Subjecting this solution to ultracentrifugation (Centricon 30) allowed the estimation of a KD of 137 µM for binding of oxidized FMN to the enzyme at room
temperature (Table II). This result
indicates the weak binding of the oxidized co-factor under these
conditions and accounts for the isolation of the protein in the
apoenzyme form and is akin to what has been observed with E. coli chorismate synthase (20). A gradual increase of the
temperature of the chorismate synthase-oxidized FMN solution to
66 °C led to a hypsochromic shift of the absorption maximum at 372 to 360 nm and was accompanied by a hypochromic effect on the absorption
intensity in this region (Fig. 6, panel B). Albeit to a
lesser degree, the increase in temperature also produced an increase in
the resolution of the absorption maximum around 450 nm and induced a
slight hypsochromic shift of the absorption maximum in this region from
445 to 442 nm (Fig. 6, panel B). The KD
for the binding of oxidized FMN to the chorismate synthase after this
heating step was estimated to be 3 µM, indicating 46-fold
tighter binding.

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Fig. 6.
UV-visible absorbance spectra of T. maritima chorismate synthase. A, absorbance
spectrum of 28 µM chorismate synthase ( ), 25 µM free oxidized FMN (- - -), chorismate synthase (28 µM), and oxidized FMN (25 µM) at 28 °C
(-·-·-·). The inset shows the absorbance spectra
amplified between 300 to 600 nm. B, absorbance spectrum of
28 µM chorismate synthase and 25 µM
oxidized FMN at 28 °C ( ), after heating to 66 °C (- - -);
after heating and addition of 108 µM EPSP (-·-·-·).
The inset shows the absorbance changes at 383 nm as a
function of the concentration of EPSP.
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Table II
Dissociation constants (KD) of T. maritima chorismate synthase
for oxidised FMN and EPSP at 28 °C
The dissociation constants for oxidised FMN were estimated in the
presence and absence of EPSP after subjecting the solution to
centrifugation in Microcon concentrators (YM-30) at 28 °C and with
and without heating to 66 °C. The dissociation constants for EPSP
were estimated from the change in absorbance at 383 nm as a function of
the concentration of EPSP under the same conditions as for oxidised FMN
using the software Sigma Plot (Jandel Scientific).
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The addition of EPSP to the heated chorismate synthase/FMNox solution
causes spectral changes which are predominantly manifested in the
450-nm absorption maximum only (Fig. 6, panel B). The main effect is an amplified resolution of the absorption maximum in this
region compared with the heating step and a bathochromic shift of the
maximum from 442 to 447 nm (Fig. 6, panel B). The spectral
changes are highlighted by observing the difference spectra as shown in
Fig. 7. From this figure it can be seen
that the temperature increase mainly affects the near UV region (Fig.
7A), while the changes observed upon the addition of the
substrate are mainly in the visible region of the spectrum,
respectively (Fig. 7B). The spectral changes occurring on
heating a solution of T. maritima chorismate synthase/FMNox
and EPSP (Fig. 7C) were the same as the sum of the changes
which occurred upon heating and then adding the EPSP (Fig.
7D). Moreover and importantly, the spectral changes observed
due to either temperature and/or adding EPSP were not reversed by
cooling. Heating free FMNox from 28 to 66 °C produced a slight
decrease in the intensity of the absorption maxima at 372 and 445 nm
(data not shown), but the changes were insignificant compared with
those observed in the presence of the protein and EPSP. The presence of
EPSP slightly decreases the KD of oxidized FMN at
28 °C and after the heat treatment, respectively (Table II). We also
estimated an apparent KD for the binding of EPSP to
the enzyme in the presence of oxidized FMN from the absorbance changes
at 383 nm as a function of the concentration of EPSP (Fig. 6,
panel B, inset). The apparent KD
estimated for EPSP (3 µM) from these experiments
decreases 10-fold upon heat treatment of the enzyme (Table II).

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Fig. 7.
Difference absorbance spectra of T. maritima chorismate synthase. A,
difference spectrum between chorismate synthase (28 µM)
and oxidized FMN (25 µM) at 66 °C and 28 °C;
B, difference spectrum between chorismate synthase/oxidized
FMN at 66 °C in the presence and absence of EPSP; C,
difference spectrum between chorismate synthase/oxidized FMN/EPSP at
66 °C and that at 28 °C; D, composite of A + B.
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Characterization of the Flavin Intermediate--
In 1991 Ramjee
et al. (21) reported on the detection (using stopped-flow
spectrophotometry) of a transient flavin intermediate formed during the
catalytic cycle of E. coli chorismate synthase and
exclusively associated with the binding of the substrate EPSP. Further
studies showed that this intermediate occurs before the substrate EPSP
is actually consumed (i.e. before C-O and C-H bond cleavage)
(22). Complementary studies showed that in the absence of substrate and
under the conditions used, pH 7.0, the reduced flavin is bound to the
protein in its deprotonated or monoanionic state
(pKa of N1-H = 6.7) (20), while in
the presence of substrate the reduced flavin is bound in its protonated
or neutral form (23). Hence, the occurrence of the flavin intermediate reflects the protonation of the deprotonated reduced flavin upon binding of substrate. As this has been one of the key observations in
the investigation of the mechanism of chorismate synthase, we performed
the same study here using rapid reaction spectrophotometry and the
T. maritima enzyme. The difference in absorption observed between a solution of T. maritima chorismate synthase/FMNred
in the presence and absence of EPSP, respectively, are shown in Fig. 8. From the inset in Fig. 8,
it can be seen that in the presence of substrate there is a rapid
increase in the absorbance at 390 nm which is complete within the dead
time of the instrument (10 ms) followed by a quasi-steady state phase
before the absorbance decreases to its initial value before adding
EPSP. From the series of difference spectra at the indicated times it
can be seen that maxima are reached at 319, 390, and 476 nm after 200 ms which decrease with time to their initial value (6 s) with
isosbestic points at 359 and 349 nm, thus indicating the homogeneity of
this species (Fig. 8). These observations are akin to those reported for the E. coli enzyme (21, 22).

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Fig. 8.
Single turnover experiment with T. maritima chorismate synthase. An anaerobic solution of
chorismate synthase (65 µM) and FMN (65 µM,
reduced with excess sodium dithionite) was mixed with EPSP (52 µM) in the stopped-flow instrument. The spectra show the
difference in absorbance detected in the presence and absence of EPSP
and were recorded at the times indicated. The inset shows
the absorbance changes at 390 nm as a function of time. The experiment
was performed at 25 °C in 10 mM HEPES, pH 7.4, containing 50 mM ammonium sulfate.
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DISCUSSION |
To our knowledge, chorismate synthase has not been purified to
homogeneity and characterized from any extremophilic organism until
now. Characterization of the enzyme was aided by cloning and expressing
the enzyme in an E. coli system which resulted in a yield of
T. maritima chorismate synthase suitable to enable the
observations reported in this study. The purification procedure is
quick and relatively easy, the greatest refinement being achieved by
the heat treatment step at 75 °C which removes almost 80% of contaminating proteins as judged from SDS-PAGE analysis. The
monofunctionality of T. maritima chorismate synthase from
the evolutionary standpoint of chorismate synthases is very
interesting. While it has been concluded from an earlier phylogenetic
analysis that chorismate synthases are monophyletic (12), it is not
known to date if the ancestral chorismate synthase is mono- or
bifunctional. However, it has been suggested that the common ancestor
was probably bifunctional given that it is difficult to imagine the
evolution of the intrinsic reductase activity in a framework of
monofunctional enzymes (12). It was surmised that bifunctionality may
have either been maintained only in organisms in which the availability
of reduced flavin is limiting or perhaps there was positive selection
of monofunctionality (12). T. maritima is thought to be one
of (if not) the oldest eubacterium and appears to have undergone
considerable lateral gene transfer from the archaea (14). A
phylogenetic tree of all chorismate synthases presently known (data not
shown) suggests that T. maritima chorismate synthase
diverged with the archaea and moreover, considerably before any of the
chorismate synthases for which bifunctionality is known
(i.e. N. crassa and Saccharomyces cerevisiae). Thus the classification of its chorismate synthase as
monofunctional could be considered to be cognate to the ancestral chorismate synthase and would therefore not lend support to
bifunctionality being ascendant.
This is the first report on the thermal denaturation behavior of any
chorismate synthase. For the mesophilic E. coli enzyme the
4 °C increase in the melting temperature in the presence of ligands
appears to be related to the major conformational change in E. coli chorismate synthase in the presence of FMN and EPSP to a more
compact structure (19). As the melting temperature of T. maritima chorismate synthase could only be estimated under the
conditions used here it was not possible to ascertain the effect (if
any) on ligand binding. However, obviously and as would be expected,
the melting temperature (92 °C) is higher than the optimal
temperature for growth of the organism (80 °C).
The quaternary structure of T. maritima chorismate synthase
is represented by a tetramer of identical subunits. This appears to
"fit" with the known quaternary structures of other chorismate synthases in that the hierarchy is spread between that of
dimer-tetramer (12). Interestingly at the quaternary level, a
remarkable feature of hyperthermophiles is the occurrence of anomalous
states of association and fused multifunctional proteins (24). However, even though there are many examples for these features (24), T. maritima chorismate synthase does not appear to be one of them. Therefore, additional quaternary interactions cannot account for the
higher stability of this protein. This question may be answered in the
future by performing specific mutations in the contact surface of the
subunits to establish their contribution to the quaternary structure.
From the native-PAGE studies performed here it could be concluded that
the apoprotein from T. maritima appears to have a higher structural rigidity compared with that of the E. coli
apoprotein which is reflected by the sharp band of the former compared
with the rather diffuse band of the latter on native-PAGE. The change in the mobility of E. coli chorismate synthase in the
presence of ligands has previously been interpreted to reflect less
conformational flexibility than that of the apoprotein (19). In support
of this, the decrease in mobility of T. maritima chorismate
synthase in the presence of ligands is not as pronounced as that
observed with the E. coli enzyme which may reflect the lower
flexibility of the thermophilic apoprotein compared with that of the
mesophilic enzyme.
In aqueous solution, the near UV-visible absorption spectra of free
flavins exhibit two featureless bands at about 450 and 375 nm (25).
However, in solvents less polar than water the band at 375 nm shifts to
a shorter wavelength and the visible band at about 450 nm shows greater
resolution with two pronounced shoulders which are thought to be
associated with vibrational transitions in the first electronic
absorption at this wavelength (26, 27). Additionally, it has been shown
that the spectra of many flavoproteins are akin to that observed for
free flavin in an apolar solvent indicating such an environment of the
flavin cofactor when bound to the protein. Importantly, most
flavoproteins show a shift in the 370 nm region to a shorter wavelength
and a varying resolution of the band at about 450 nm (25). With T. maritima chorismate synthase, an apolar type flavin
spectrum is observed when the temperature of the protein/FMN solution
is increased, thus reflecting induced FMN binding. This is borne out by
the fact that the KD for FMN binding is ~46-fold lower after heating to 66 °C and cooling to 28 °C compared with that measured at 28 °C without heating to 66 °C (3-137
µM, respectively). Upon FMN binding, the spectral
transition is mainly manifested in the 370-nm region which is
consistent with solvent-dependent spectral shifts (more
apolar) with little development of fine structure. This is accompanied
by slightly better resolution in the 450-nm region which indicates that
the vibronic transitions in this area are more discrete. This is
clearly due to binding of the flavin to the protein which reduces the
rotational freedom of the flavin and possibly introduces some strain on
the bound cofactor. The phenomenon is further enhanced by the presence
of EPSP which substantially increases the band resolution in this area
and thus probably the constraint on the flavin. This observation may
reflect that T. maritima chorismate synthase has a more
rigid/compact structure at room temperature which upon heating becomes
more flexible resulting in an increase in the rate of ligand binding and in fact probably allows the cofactor to enter into the active site.
This view is supported by the fact that cooling the protein/FMN solution to room temperature after heating does not reverse the process, indicating that the FMN is now "trapped" in the active site.
The flavin-derived transient intermediate previously observed for the
E. coli enzyme (21) was also observed in this study with
T. maritima chorismate synthase. As for the E. coli enzyme, formation of this flavin species was associated with
binding of the substrate, i.e. formation of a ternary
complex between enzyme, reduced flavin, and EPSP. Formation of this
complex is considered to reflect protonation of the anionic reduced
flavin, at least for the E. coli enzyme (20, 23). This
protonation of the reduced flavin indicates that the flavin experiences
a different polarity in the active site when substrate binds to the
enzyme and is supported by the apolar-type flavin spectra observed here
when EPSP is added to T. maritima chorismate synthase/FMNox
after heating. The observation suggests that the catalytic action of
T. maritima chorismate synthase proceeds with a mechanism
similar to that of E. coli chorismate synthase,
i.e. flavin-derived intermediate formation (reduced FMN in
neutral form) precedes C-O and C-H bond cleavage of EPSP (22).
Finally, one of the major drawbacks at present in proceeding with
studies on this intriguing enzyme is the lack of a three-dimensional structure. For x-ray crystallography, it has been suggested that adequately diffracting crystals can be obtained more readily with thermophilic enzymes due to their greater rigidity and stability (28).
The apparent rigidity and stability of the protein described here
coupled with the characteristics of substrate binding make it an
exceptional source with regard to the possibility of elucidating the
structure of chorismate synthase.