Although no net redox change occurs during the reaction, chorismate synthase activity is based on the supply of reduced FMN, which is bound in the active site of the enzyme [3–5].. In th
Trang 1chorismate synthase provides evidence that a proton relay system is essential for intermediate formation and
catalytic activity
Gernot Rauch1, Heidemarie Ehammer1, Stephen Bornemann2and Peter Macheroux1
1 Institute of Biochemistry, Graz University of Technology, Austria
2 Department of Biological Chemistry, John Innes Centre, Norwich, UK
Chorismate synthase catalyzes the seventh and last step
in the shikimate pathway, leading to chorismate, the
last common precursor in the biosynthesis of
numer-ous aromatic compounds in bacteria, fungi, plants and
protozoa Because of the absence of this pathway in
eukaryotic organisms, its enzymes are interesting
potential targets for rational drug design [1] The
chorismate synthase reaction involves an
anti-1,4-elimi-nation of the 3-phosphate and the C(6proR) hydrogen,
as shown in Scheme 1 [2,3]
Although no net redox change occurs during the reaction, chorismate synthase activity is based on the supply of reduced FMN, which is bound in the active site of the enzyme [3–5] Mechanistic studies have indi-cated a functional role of the reduced flavin [6,7] that comprises the transient donation of an electron (or a
Keywords
enzyme mechanism elimination; flavin;
shikimate pathway; site-directed
mutagenesis
Correspondence
P Macheroux, Institute of Biochemistry,
Graz University of Technology,
Petersgasse 12 ⁄ II, A-8010 Graz, Austria
Fax: +43-316-873 6952
Tel: +43-316-8736450
E-mail: peter.macheroux@tugraz.at
(Received 6 December 2007, revised 15
January 2008, accepted 21 January 2008)
doi:10.1111/j.1742-4658.2008.06305.x
Chorismate synthase is the last enzyme of the common shikimate pathway, which catalyzes the anti-1,4-elimination of the 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate (EPSP) to generate chorismate, a precursor for the biosynthesis of aromatic com-pounds Enzyme activity relies on reduced FMN, which is thought to donate an electron transiently to the substrate, facilitating C(3)–O bond breakage The crystal structure of the enzyme with bound EPSP and the flavin cofactor highlighted two invariant serine residues interacting with a bound water molecule that is close to the C(3)–O of EPSP In this article
we present the results of a mutagenesis study where we replaced the two invariant serine residues at positions 16 and 127 of the Neurospora crassa chorismate synthase with alanine, producing two single-mutant proteins (Ser16Ala and Ser127Ala) and a double-mutant protein (Ser16Ala-Ser127Ala) The residual activity of the Ser127Ala and Ser16Ala single-mutant proteins was found to be six-fold and 70-fold lower, respectively, than that of the wild-type protein No residual activity was detected for the Ser16AlaSer127Ala double-mutant protein, and formation of the typical transient intermediate, characteristic for the chorismate synthase-catalysed reaction, was not observed, in contrast to the single-mutant proteins On the basis of the structure of the enzyme, we propose that Ser16 and Ser127 form part of a proton relay system among the isoalloxazine ring of FMN, histidine 106 and the phosphate group of EPSP that is essential for the for-mation of the transient intermediate and for substrate turnover
Abbreviations
EPSP, 5-enolpyruvylshikimate 3-phosphate; NcCS, Neurospora crassa chorismate synthase; wat 1 wat 2 and wat 3, water molecules in chorismate synthase.
Trang 2charge transfer) to the substrate, prompting cleavage
of the C–O bond and thereby facilitating phosphate
cleavage At the end of the catalytic cycle an electron
(or negative charge) is redistributed to maintain the
reduced form of the flavin cofactor [8–11]
The theoretical and experimental evidence for such a
role of the reduced FMN eagerly demanded structural
information of the protein Eventually, the structure
determination of Streptococcus pneumoniae chorismate
synthase in the presence of oxidized FMN and
5-enol-pyruvylshikimate 3-phosphate (EPSP) provided the first
insight into the binding and relative orientation of the
cofactor and of the substrate in the active site of the
enzyme [10] Based on this structure we were able to
initiate a structure-based mutagenesis study to test
mechanistic proposals In our first study we
demon-strated that the invariant histidine residues (His17 and
His106) function as general acids in the active site, with
His106 protonating the N(1)–C(2)=O locus of reduced
flavin whereas His17 appears to be involved in the
pro-tonation of the leaving phosphate group [12] The next
target was Asp367, which is in the direct vicinity of the
N(5) atom of the isoalloxazine ring system and a likely
candidate in a position for the abstraction of hydrogen
from the substrate Single-mutant proteins in which
Asp367 has been replaced with alanine or asparagine
exhibit a 300- and 600-fold lower activity, respectively,
emphasizing the important role of the Asp367 residue
as an active-site base These results provide strong
evi-dence that acid–base catalysis is of great importance in
the chorismate synthase reaction [13]
Multiple sequence alignments of chorismate
synthas-es from bacterial, fungal, plant and protozoan origin,
of the crystal structure of the enzyme with bound
EPSP and of the flavin cofactor, revealed two invariant
serine residues – Ser16 and Ser127 – interacting with
several bound water molecules As shown in Fig 1,
one water molecule (wat 1) is held by both serine side
chains (in the reported structure of S pneumoniae
chorismate synthase, these positions are designated as
Ser9 and Ser132, [10]) close to the C(3)–oxygen, while
another water molecule (wat 2) is bound between a
third water molecule (wat 3) and a C1 carboxyl oxygen
of the substrate that is also hydrogen bonded to
His106 The third water molecule bridges the first two
water molecules There are both open (Fig 1A) and closed (Fig 1B) active-site structures that reveal a tightening up of the site in the latter together with a movement of the His106 side chain away from the fla-vin and towards the substrate [its ring nitrogen atom that participates in hydrogen bonding moves 1.3 A˚ away from the C(2=O) oxygen of the flavin ring sys-tem and 1.2 A˚ closer to the oxygen atom of the car-boxyl group of EPSP]
In order to probe the pertinent role of the Ser16 and Ser127 residues, we generated two single-mutant pro-teins where the two serine residues were replaced with alanine, producing two single-mutant proteins (Ser16-Ala and Ser127(Ser16-Ala) and a double-mutant protein (Ser16AlaSer127Ala) In this article, we report that the replacement of both invariant serine residues (Ser16AlaSer127Ala double-mutant protein) in the active site of chorismate synthase caused a substantial decrease in activity beyond the detection limit of our assay In contrast to the single-mutant proteins (Ser16Ala and Ser127Ala) the Ser16AlaSer127Ala dou-ble-mutant protein was not able to form the typical transient intermediate Based on our results, we pro-pose that Ser16 and Ser127 establish a proton relay system among the isoalloxazine ring, His106 and the EPSP molecule that delivers protons via water mole-cules either to His106, protonating the flavin at N(1) position, or to the C(3)–oxygen of the phosphate-leaving group to facilitate C–O bond breakage
Results
Expression and purification of the Ser16Ala and Ser127Ala single-mutant proteins and of the Ser16AlaSer127Ala double-mutant protein The mutant proteins were heterologously expressed
in Escherichia coli, strain BL21(DE3)RP at expression levels similar to those of the wild-type protein The two-step chromatographic procedure developed for the purification of wild-type enzyme yielded similar amounts of the mutant proteins (3 mg of protein per gram of wet cell paste) [14] Because of the weak bind-ing of (oxidized) FMN, all mutant proteins were iso-lated in their apo-form The apparent stability of all three mutant proteins was comparable to that of the wild-type enzyme
Binding of oxidized FMN to the mutant proteins
To characterize the serine mutant proteins in further detail, binding of the oxidized FMN cofactor to the isolated apoproteins was investigated by UV⁄ visible
Scheme 1 Reaction catalyzed by chorismate synthase.
Trang 3difference absorbance spectroscopy (Fig 2) The
spec-tral changes observed upon binding of oxidized FMN
to the serine mutant proteins were identical to those
seen with the wild-type enzyme [14] The dissociation
constant for the Ser127Ala mutant protein (Table 1)
was similar to that of the wild-type enzyme, whereas
the replacement of Ser16 with alanine resulted in a
slight increase of the dissociation constant (inset of
Fig 2 and Table 1) Similarly, the Ser16AlaSer127Ala
double-mutant protein showed slightly weaker binding
of the cofactor (Table 1) Note that for the wild-type
E coli protein, its dissociation constant for flavin
decreases by three orders of magnitude when the flavin
becomes reduced [15]
Binding of EPSP to the mutant proteins in the
presence of oxidized FMN
Because the replaced serine residues are located in the
direct vicinity of EPSP (Fig 1A) it is important to
ensure that binding of EPSP to the active site is not hampered Binding of EPSP to the Ser16Ala, Ser127-Ala and Ser16Ser127-AlaSer127Ser127-Ala mutant proteins in the presence of oxidized FMN was monitored by UV⁄ visi-ble spectroscopy The spectral changes observed upon the binding of EPSP were exploited to determine the dissociation constants for EPSP The spectral perturba-tions on EPSP binding, and the calculated dissociation constants for the Ser16Ala and the Ser127Ala single-mutant proteins, were found to be similar to those of the wild-type enzyme (Table 1) As shown in Fig 3, the spectral changes observed when EPSP bound
to the double-mutant protein were comparable to those observed with the wild-type protein, whereas the calculated dissociation constant was 10-fold higher than observed with the wild-type enzyme [14] Note that the Km for EPSP was 2.7 lm with the wild-type enzyme [16] and therefore one order of magnitude lower than the dissociation constant determined in the presence of oxidized flavin
B
A
Fig 1 Ser16 and Ser127 residues acting in
a proton relay system among His106, FMN and EPSP via a water molecule in the chorismate synthase active site (A) Stereo-representation of the active site in the open form, where His106 is in a position close to C(2)=O of the flavin (B) Stereorepresenta-tion of the chorismate synthase active site
in the closed form, where His106 makes contact with the O12 of EPSP The carbon atoms of Ser16, Ser127, His106, FMN and EPSP are colored gray The red spheres rep-resent water positions Hydrogen bonds are shown as dashed lines wat 1, wat 2, wat 3, are different water molecules.
Trang 4Fig 2 Binding of oxidized FMN to the
Ser16Ala mutant protein The plot shows
the result of titration of the Ser16Ala mutant
protein (23 l M ) with oxidized FMN in 50 m M
Mops buffer, pH 7.5 Arrows indicate the
direction of the spectral changes occurring
upon titration with FMN Difference
absor-bance spectra at 0, 9.8, 14.6, 22.3, 41.2 and
78.1 l M oxidized FMN are shown The inset
shows the spectral changes at 379 nm as a
function of FMN concentration, revealing a
dissociation constant (Kd) of 60 l M
Table 1 Dissociation constant (K d ) values for FMN and EPSP.
Ligand
K d (l M )
Method Wild-type
NcCS
NcCS Ser16Ala
NcCS Ser127Ala
NcCS Ser16Ala Ser127Ala
EPSP
(in the presence of FMN)
a Average of three independent measurements b Average of two independent measurements.
Fig 3 Binding of EPSP to the
Ser16Ala-Ser127Ala double-mutant protein in the
presence of oxidized FMN The course of a
titration of the Ser16AlaSer127Ala
double-mutant protein with EPSP in 50 m M Mops
buffer, pH 7.5 is shown UV-visible
absor-bance spectra of the Ser16AlaSer127Ala
double-mutant protein (30 l M ) and FMN
(25 l M ) were recorded at various EPSP
con-centrations The spectra shown are at the
following EPSP concentrations: 0, 5.7, 9.9,
21, 42.4, 69.4, 109.3, 148.6, 200 and
250.3 l M The arrows indicate the direction
of the absorbance changes The inset
shows the spectral changes at 397 nm as a
function of EPSP concentration, revealing a
dissociation constant (Kd) of 155 l M
Trang 5Intrinsic FMN:NADPH oxidoreductase activity
of the mutant proteins
Chorismate synthase from Neurospora crassa has an
intrinsic NADPH:FMN oxidoreductase activity that
enables the enzyme to generate the reduced FMN
cofactor (bifunctionality) The structural basis of this
‘secondary’ catalytic activity is presently not known
[4,17] We investigated the effect of the mutations
on the NADPH:FMN oxidoreductase activity of the
mutant proteins From the obtained hyperbolic
depen-dency, Michaelis–Menten parameters of 14, 14, 4 and
7 lm were calculated for the wild-type protein and for
the Ser16Ala, Ser127Ala and Ser16AlaSer127Ala
mutant proteins, respectively These results
demon-strated that none of the amino acid replacements
significantly affected the utilization of NADPH as a
source of reducing equivalents for activation of the
cofactor to its reduced form
Chorismate synthase activity of the serine
mutant proteins
In order to investigate the influence of the amino acid
replacements on the catalytic activity of chorismate
syn-thase, we measured the activity of the mutant proteins
An activity assay under aerobic conditions using
NADPH as a source of reducing equivalents (15)
indi-cated that the amino acid replacements have a large
effect on the activity of the mutant proteins in
com-parison to the wild-type enzyme Under these
condi-tions, we measured a residual activity of 2% for the
Ser16Ala single-mutant protein and of 12% for the
Ser127Ala single-mutant protein compared with that
of the wild-type enzyme However, we were not able
to determine any activity for the Ser16AlaSer127Ala
double-mutant protein
The precise residual activity of the mutant proteins
was then measured using the stopped-flow instrument
under anoxic conditions where we used photoreduction
to generate the reduced flavin cofactor In the absence
of oxygen, the rate of chorismate formation was six- and
70-fold lower for the Ser127Ala and Ser16Ala mutant
proteins, respectively, than for the wild-type enzyme, as
shown in Table 2 The residual activity of the
Ser16Ala-Ser127Ala double-mutant protein was below the
detec-tion limit of the instrument (Table 2) Therefore, we
performed the same activity assay with a 10-fold higher
concentration of the Ser16AlaSer127Ala mutant protein
(125 lm instead of 12.5 lm) but again we were unable
to detect chorismate formation
The chorismate synthase-catalysed reaction is
char-acterized by the occurrence of a transient species
with an absorbance maximum at around 390 nm [18] This species is known to form after the sub-strate binds to the reduced FMN–enzyme binary complex [9] but before EPSP undergoes transforma-tion to the product [18,19] This species is formed very rapidly (within a few milliseconds) and dis-appears when all substrate has been consumed The formation of the intermediate by the two serine single-mutant proteins was almost complete within the dead time of the instrument and indistinguishable from that of the wild-type in single-turnover experi-ments (Fig 4) For the Ser16AlaSer127Ala double-mutant protein we were not able to detect the intermediate This demonstrates that the Ser16Ala-Ser127Ala double-mutant protein, in contrast to the single-mutant proteins (Ser16Ala and Ser127Ala) is not capable of forming the flavin-derived intermedi-ate The decay rate of the intermediate in a single-turnover experiment in general reflects the rate of substrate turnover In the case of the Ser127Ala and Ser16Ala single-mutant proteins, the decay of the transient species was eight- and 140-fold slower, respectively, than that of the wild-type enzyme, in good agreement with the slower rate of substrate turnover (Table 3)
In contrast to the wild-type protein, the spectra of the transient flavin species of the two single serine mutant proteins (Ser16Ala and Ser127Ala) have slightly different spectral properties As shown in Fig 5, both single-mutant proteins show, in addition
to the peak at 390 nm, a broad shoulder in the range
of 430–480 nm Thus, both serine single-mutant pro-teins (Ser16Ala and Ser127Ala) affect the spectral characteristics of the transient flavin intermediate Sim-ilar spectral changes were observed during turnover with the substrate analogue (6S)-6-fluoro-EPSP [20]
Table 2 Chorismate synthase activity of the serine mutant pro-teins (Ser16Ala, Ser127Ala and Ser16AlaSer127Ala) in comparison with the activity of the wild-type enzyme The formation of choris-mate was monitored at 275 nm using stopped-flow spectrophoto-metry under anaerobic conditions.
Chorismate synthase activity
NcCS Wild-type
NcCS Ser16Ala
NcCS Ser127Ala
NcCS Ser16Ala Ser127Ala
detection limit
a Chorismate synthase activity compared with wild-type NcCS activity.
Trang 6Thus, small perturbations in the substrate and its
immediate vicinity have similar effects on the flavin
environment
Discussion
The 1,4-elimination of the 3-phosphate group and the C-(6proR) hydrogen from EPSP to chorismate by chorismate synthase is still one of the most challeng-ing flavin-dependent reactions The activity of the chorismate synthase-catalysed reaction is dependent
on the supply of reduced FMN, which is bound in the active site of the enzyme [3–5,10] Several kinetic and mechanistic studies have accumulated substantial evidence for a radical mechanism in which the enzyme-bound reduced FMN facilitates C–O bond cleavage by transient electron donation (or negative charge transfer) to the substrate [6,7] At the end of the catalytic cycle, an electron (or negative charge) is redistributed to maintain the reduced form of the
Fig 4 Formation of a transient flavin intermediate during substrate
turnover with wild-type enzyme (A), the Ser127Ala mutant protein
(B) and the Ser16Ala mutant protein (C) The absorbance changes
were observed at 390 nm as a function of time in single-turnover
experiments under anoxic conditions using stopped-flow
spectro-photometry The formation of the intermediate was obscured by
the dead time of the instrument but its exponential decay is clearly
visible.
Table 3 Decay rates of the transient flavin intermediate The decay rates for the wild-type NcCS and for the two single-mutant proteins were obtained using stopped-flow spectrophotometry under anaerobic conditions (single turnover) The absorbance changes were observed at 390 nm as a function of time.
NcCS Wild-type
NcCS Ser16Ala
NcCS Ser127Ala
NcCS Ser16Ala Ser127Ala
Decay rate (s)1)
detected
a Chorismate synthase decay rates compared with the wild-type NcCS decay rate.
Fig 5 Observation of the flavin intermediate in the chorismate synthase reaction Comparison of the difference absorbance spec-tra formed during the reaction with wild-type enzyme (solid line), the Ser16Ala mutant protein (dotted line) and the Ser127Ala mutant protein (dashed line) The wild-type (Wt) trace was taken from Kitzing et al [12] The spectra were obtained during a multiple-turnover experiment, and those obtained with the highest ampli-tude within the first few seconds after mixing are shown Spectra decayed with time but did not otherwise change.
Trang 7flavin cofactor [8–11] The elucidation of the
3D structure of the S pneumoniae chorismate
syn-thase in the presence of oxidized FMN and EPSP
(catalytically inactive ternary complex) has provided
the first insight into the binding and relative
orienta-tion of the cofactor and the substrate in the active site
of the enzyme [10] A structure of the catalytically
active ternary complex among enzyme, substrate and
reduced FMN would be difficult to obtain because it
would turn over to give the product, for which the
enzyme has a much poorer affinity The structure of
the active site of chorismate synthase is consistent with
the hitherto proposed role of reduced FMN, as
out-lined above, and also reveals several invariant amino
acid residues in the active site of the enzyme Based on
this structural information we performed our first
mutagenesis study where we investigated the role of
two conserved histidine residues (His17 and His106),
revealing their role as general acid–base catalysts [12]
Recently, we reported experimental evidence that an
invariant aspartate residue (Asp367) operates in
con-cert with N(5) of the cofactor to bring about the
abstraction of the C(6proR) hydrogen of the substrate
[13] In addition to these invariant amino acid residues,
the active site of chorismate synthase features two
strictly conserved (99.5% of 400 sequences aligned)
serine residues located near the substrate on the
oppo-site oppo-site of the isoalloxazine ring (Fig 1A) From this
structure the functional role of the serine residues is
not obvious although it was speculated that the side
chains help to organize the water molecules in the
active site [10] As shown in Fig 1, one of these water
molecules (wat 1) hydrogen bonds to the C(3)–oxygen
of the phosphate group and is held by both serine
residues, while another water molecule (wat 2) is
positioned between a third water molecule and a
C1 carboxyl oxygen of the substrate that is also
hydro-gen bonded to His106 The third water molecule
(wat 3) links the first two water molecules This
inter-esting configuration in the active site of chorismate
synthase, which seems to constitute a proton relay
sys-tem among the isoalloxazine ring of FMN, histidine
106 and the EPSP molecule, prompted us to
investi-gate the role of Ser16 and Ser127 for the chorismate
synthase-catalysed reaction
First, we analyzed the ability of the mutant proteins
to bind cofactor (Fig 2) and substrate (Fig 3) All
three serine mutant proteins were able to bind oxidized
FMN with dissociation constants comparable to that
of the wild-type enzyme but we observed a significant
difference in the ability to bind EPSP, as expected
While the serine single-mutant proteins (Ser16Ala and
Ser127Ala) have EPSP dissociation constants similar
to those of the wild-type enzyme, the dissociation con-stant for the Ser16AlaSer127Ala double-mutant pro-tein was 10-fold higher than for the wild-type enzyme (Table 1) As shown in Fig 1A, the Ser16 and Ser127 residues stabilize a water molecule (wat 1), which forms a hydrogen bond to the C(3)-oxygen of EPSP Moreover, wat 2 forms a hydrogen bond to the car-boxylate group of EPSP Therefore, we conclude that the entire hydrogen bonding network is disrupted in the double-mutant protein, as indicated by the 10-fold higher dissociation constant for EPSP binding (Fig 3 and Table 1)
Next, we studied the effects on the catalytic proper-ties of our mutant proteins Both single-mutant pro-teins showed a modest to strong decrease in activity by factors of 6 and 70 for the Ser127Ala and the Ser16-Ala mutant proteins, respectively (Table 2) This is also probably a result of the loss of appropriately ordered water within the EPSP-binding site It is possi-ble that the Ser16Ala mutation is more disruptive because it might also affect the orientation of its neighbour, His17, which normally hydrogen bonds to the phosphate-leaving group Most importantly, the Ser16AlaSer127Ala double-mutant protein is devoid
of any detectable catalytic activity, indicating that replacement of both serine residues produces a syner-gistic effect This result is consistent with the inability
of the double-mutant protein to form the transient flavin intermediate, which normally forms before any bond-breaking steps occur [18,19] Taken together, our results suggest that the absence of one serine residue leads to a partial disruption of the water structure (‘conserved water molecules’), whereas the absence of both serine residues generates an environment that abrogates the required water structure
Therefore, we propose a mechanism in which a pro-ton is relocated from the N(1)–C(2)=O locus of the isoalloxazine ring to the imidazole ring of His106, then shuttled to the phosphate ester oxygen atom of the phosphate-leaving group, a process mediated by wat 1, wat 2, wat 3 and the carboxyl group of EPSP, which serve as a proton translocation system in the active site (see Fig 1B and Scheme 2) Interestingly, the proton
on the N(1)–C(2)=O locus is likely to have originally come from His106 The transient flavin intermediate is thought to be the result of the protonation of anionic reduced flavin on binding of EPSP to give neutral reduced flavin [9], and the associated general acid is thought to be His106 [10] It is therefore possible that disruption of the proposed proton relay system affects not only phosphate cleavage, but also this first flavin-protonation step, by affecting the initial flavin-protonation state of His106
Trang 8Such a proton relay system is attractive for several
reasons The enzyme initiates catalysis by ‘separation’
of an electron and a proton, both of which are derived
from the reduced protonated flavin in a
proton-cou-pled electron transfer step The electron is donated to
the substrate in order to facilitate C–O bond-breakage
Phosphate dianions are poor leaving groups, and
although interactions with His10, Arg49 and Arg337
facilitate the neutralization of the negative charge on
the phosphate group of the substrate, a mechanism for
lowering the incipient negative charge on the oxygen
of the CO bond being cleaved would be expected
Upon product dissociation, this internal proton relay
system can be reloaded, leading to protonation of
His106, such that the enzyme is ready for the next
sub-strate turnover
In this mechanism, His106 plays a central role,
supported by structural evidence that this residue has
some conformational flexibility allowing it to assume
different positions in the active site [10] In the
so-called open conformation (Fig 1A), His106 assumes
a position close to the C(2)=O position of the
isoallox-azine ring system, whereas in the closed conformation
(Fig 1B), it moves away from the flavin towards the
substrate and makes contact with an oxygen atom
(O12) of the substrate’s carboxylate group, which, in
turn, is in hydrogen bond distance to wat 2 Hence, it
appears that His106 and the substrate’s carboxylate
group function as a gate, controlling proton transfer in
the enzyme active site during catalysis Furthermore,
the tightening of the active site in the closed structure
is required for a hydrogen bond to form between
wat 1 and wat 3, adding another element to such a
gating mechanism In summary, our data provide
evi-dence that the two invariant serine residues are
required to organize a chain of water molecules in the
active site of chorismate synthase, which form a
pro-ton relay system among the isoalloxazine ring of
FMN, His106 and substrate This proton relay system
is essential for catalysis and is probably synchronized with the electron transfer process to the substrate, emphasizing the unique character of the chorismate synthase reaction
Experimental procedures
Reagents All chemicals were of the highest grade available and obtained from Sigma or Fluka (Buchs, Switzerland) DEAE Sephacel was from Amersham Biosciences, and cellulose phosphate (P11) was from Whatman (Kent, UK) DNA restriction and modification enzymes were obtained from Fermentas GmbH or from New England Biolabs (Beverly,
MA, USA) Plasmid DNA preparation was performed using the Nucleobond AX plasmid preparation kit (Mache-rey-Nagel GmbH & Co KG, Germany) PCR primers were purchased from VBC-Genomics (Vienna, Austria) EPSP was synthesized from shikimate-3-phosphate using recombi-nant E coli EPSP synthase and purified by HPLC
Site-directed mutagenesis Basic molecular biology manipulations were performed using standard techniques [21] Amino acid replacements were performed using the QuikChange site-directed muta-genesis kit from Stratagene (La Jolla, CA, USA) The construct pET21a–N crassa chorismate synthase (pET21a– NcCS) served as the template The following oligonucleo-tides containing the appropriate codon exchange were used for the procedure (the changed codons are underlined and nucleotides exchanged are in bold): S16A, forward pri-mer, 5¢-CGACCTATGGCGAGGCGCACTGCAAGTCG-3¢, and reverse primer, 5¢-CGACTTGCAGTGCGCCTCGC CATAGGTCG-3¢; S127A, forward primer, 5¢-GCGGCCG CTCTGCCGCCCGCGAGACC-3¢, and reverse primer, 5¢-GGTCTCGCGGGCGGCAGAGCGGCCGC-3¢ For the S16AS127A mutein, the construct pET21a–NcCS-S16A served as the template and the primer for the S127A
replace-CO2
OH O O
–
O 2 C
H
3 6
N
N NH H
O
O
1 5
N
N NH N
O
O Electron
transfer
H2 C N
NH His106
H 2
C N
NH His106
P O O O
NH CH C
H 2
C O
H O
O H H
Ser127 O
H H
NH CH C
H2 C O
wat1
wat2
Scheme 2 Proposed proton relay system.
Trang 9ment was used in the procedure All manipulations were
performed following the manufacturer’s instructions The
mutations were verified by DNA sequencing (MWG-Biotech
AG, Germany)
Production and purification of NcCS
The Ser16Ala, Ser127Ala and Ser16AlaSer127Ala mutant
proteins were produced and purified as described for the
wild-type enzyme [14] Concentration of the purified mutant
proteins was carried out using Ultracel YM-10 Centriprep
concentrators (Amicon Bioseparations, Bedford, MA, USA)
UV-visible absorbance spectrophotometry
Absorbance spectra for EPSP titration experiments were
recorded using a Specord 210 spectrophotometer equipped
with a thermostated cell holder (Analytik Jena, Germany)
All experiments were performed in 50 mm Mops buffer,
pH 7.5, at 25C
UV⁄ visible difference absorbance
spectrophotometry
Binding of oxidized FMN to NcCS can be directly
moni-tored by difference UV⁄ visible spectrophotometry For this
experiment, tandem cuvettes (Hellma GmbH & Co KG,
Germany) were used Initially, the first chamber in the
opti-cal path of the sample and the reference cuvette was filled
with enzyme solution, whereas the second chamber was
filled with the same volume of buffer The titration was
per-formed by successive additions of the same volume of an
FMN solution to the enzyme solution of the sample cuvette
and to the buffer compartment of the reference cuvette To
compensate for the dilution of the enzyme solution in the
reference cuvette, the same volume of buffer was added to
the enzyme solution in the reference cuvette The observed
spectral changes on binding of oxidized FMN to the
enzyme were exploited to determine the dissociation
con-stants for oxidized FMN
Activity assay under aerobic conditions
Chorismate synthase activity was determined by measuring
chorismate formation at 281 nm The reactions were started
by the addition of 30 lm EPSP to a mixture of 100 lm
NADPH, 25 lm FMN and 4 lm of either the wild-type
NcCS or the mutant proteins Reactions were carried out in
50 mm Mops, pH 7.5, at 25C [14]
Stopped-flow spectrophotometry
Single-turnover and multiple-turnover experiments were
carried out using a Hi-Tech Scientific SF-61 stopped-flow
spectrophotometer (Salisbury, UK) at 25C The dead time
of the instrument was measured to be 4.1 ± 0.3 ms [22] The stopped-flow observation cell had a 1.0 cm path length Enzyme and substrate solutions were made anaerobic by exchanging the dissolved oxygen with argon by several cycles
of evacuation and flushing Anaerobic substrate solution was rapidly mixed with anaerobic enzyme solution in the spectro-photometer Both solutions contained FMNH2 that was reduced using photoirradiation, in the presence of potassium oxalate, prior to mixing After mixing, the anaerobic reaction mixture contained reduced FMN (80 lm), EPSP (100 lm), oxalate (1 mm), 50 mm Mops, pH 7.5, and enzyme (1.25 lm for the wild-type and Ser127Ala mutant proteins
or 12.5 lm of the Ser16Ala and Ser16AlaSer127Ala mutant proteins) Chorismate formation was monitored at 275 nm under anaerobic conditions
For determination of the decay rates of the transient fla-vin intermediate for the wild-type and mutant proteins, the absorbance changes were observed at 390 nm, as a function
of time, in single-turnover experiments with 20 lm enzyme and 15 lm substrate Kinetic data were fitted using the Hi-Tech Scientific kinetasyst 3.14 software
Spectra of reaction intermediates Absorbance spectra were recorded with a Hewlett-Packard photodiode array instrument (model HP8452) using a cuv-ette with a side arm The enzyme solutions and the sub-strate in the side arm of the cuvette were made anaerobic
by exchanging the dissolved oxygen by several cycles of evacuation and flushing with argon The anaerobic enzyme solution containing FMNH2 was reduced using photoirra-diation in the presence of potassium oxalate before mixing with the substrate and recording the spectra After mixing, the anaerobic reaction mixture contained enzyme (40 lm), reduced FMN (80 lm), EPSP (80 lm) and oxalate (1 mm),
in 50 mm Mops, pH 7.5 Absorbance spectra were recorded between 300 and 600 nm Each blank was obtained from control spectra in the presence of fully reduced FMN, but
in the absence of substrate
Acknowledgements
This work was supported, in part, by the Fonds zur Fo¨rderung der wissenschaftlichen Forschung (FWF) through grant P17471 to PM
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