Replacement of two invariant serine residues in
chorismate synthase provides evidence that a proton relay
system is essential for intermediate formation and
catalytic activity
Gernot Rauch
1
, Heidemarie Ehammer
1
, Stephen Bornemann
2
and Peter Macheroux
1
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

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.
1464 FEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBS
charge 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-

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 Ser127Ala) 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

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 Ser16AlaSer127Ala 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 K
m
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

function of FMN concentration, revealing a
dissociation constant (K
d
)of60lM.
Table 1. Dissociation constant (K
d
) values for FMN and EPSP.
Ligand
K
d
(lM)
Method
Wild-type
NcCS
NcCS
Ser16Ala
NcCS
Ser127Ala
NcCS Ser16Ala
Ser127Ala
FMN 41 ± 5
a
60 ± 7
a
40 ± 9
a
56
b
UV ⁄ visible difference spectroscopy
EPSP

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 (K
d
) of 155 lM.
G. Rauch et al. Proton relay system in chorismate synthase
FEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBS 1467
Intrinsic 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

[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

a
100 1.38 16.1
a
Chorismate synthase activity compared with wild-type NcCS
activity.
Proton relay system in chorismate synthase G. Rauch et al.
1468 FEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBS
Thus, 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-

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.
G. Rauch et al. Proton relay system in chorismate synthase
FEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBS 1469
flavin 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-

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

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

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¢-CGACCTATGGCGAG
GCGCACTGCAAGTCG-
3¢, and reverse primer, 5¢-CGACTTGCAGTG
CGCCTCGC
CATAGGTCG-3¢; S127A, forward primer, 5¢-GCGGCCG
CTCT
GCCGCCCGCGAGACC-3¢, and reverse primer,
5¢-GGTCTCGCGGGC
GGCAGAGCGGCCGC-3¢. For the
S16AS127A mutein, the construct pET21a–NcCS-S16A
served as the template and the primer for the S127A replace-
CO
2
–
OH
OO
–
O
2
C
H
3
6
N
H
N

O
O
O
NH
CH
C
H
2
C
O
H
O
O
H
H
Ser127
O
H
H
NH
CH
C
H
2
C
O
HO
Ser16
wat1
wat2

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 25 °C [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 25 °C. 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 FMNH
2
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

This work was supported, in part, by the Fonds zur
Fo
¨
rderung der wissenschaftlichen Forschung (FWF)
through grant P17471 to PM.
References
1 Coggins JR, Abell C, Evans LB, Frederickson M, Rob-
inson DA, Roszak AW & Lapthorn AJ (2003) Experi-
ences with the shikimate-pathway enzymes as targets
for rational drug design. Biochem Soc Transactions 31,
548–552.
Proton relay system in chorismate synthase G. Rauch et al.
1472 FEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBS
2 Floss HG, Onderka DK & Carroll M (1972) Stereo-
chemistry of the 3-deoxy-d-arabino-heptulosonate
7-phosphate synthetase reaction and the chorismate
synthetase reaction. J Biol Chem 247, 736–744.
3 Morell H, Clark MJ, Knowles PF & Sprinson DB
(1967) The enzymic synthesis of chorismic and prephe-
nic acids from 3-enolpyruvylshikimic acid 5-phosphate.
J Biol Chem 242, 82–90.
4 Welch GR, Cole KW & Gaertner FH (1974) Choris-
mate synthase of Neurospora crassa: a flavoprotein.
Arch Biochem Biophys 165, 505–518.
5 White PJ, Millar G & Coggins JR (1988) The overex-
pression, purification and complete amino acid sequence
of chorismate synthase from Escherichia coli K12 and
its comparison with the enzyme from Neurospora
crassa. Biochem J 251, 313–322.
6 Bornemann S (2002) Flavoenzymes that catalyse reactions

chorismate synthase from Neurospora crassa. Evidence
for a common binding site for 5-enolpyruvylshikimate
3-phosphate and NADPH. J Biol Chem 276, 42658–
42666.
15 Macheroux P, Petersen J, Bornemann S, Lowe DJ &
Thorneley RNF (1996) Binding of the oxidized,
reduced, and radical flavin species to chorismate syn-
thase. An investigation by spectrophotometry, fluorime-
try, and electron paramagnetic resonance and electron
nuclear double resonance spectroscopy. Biochemistry
35, 1643–1652.
16 Balasubramanian S, Abell C & Coggins JR (1990)
Observation of an isotope effect in the chorismate
synthase reaction. J Am Chem Soc 112, 8581–8583.
17 Henstrand JM, Amrhein N & Schmid J (1995) Cloning
and characterization of a heterologously expressed
bifunctional chorismate synthase ⁄ flavin reductase from
Neurospora crassa. J Biol Chem 270, 20447–20452.
18 Bornemann S, Lowe DJ & Thorneley RNF (1996)
The transient kinetics of Escherichia coli chorismate
synthase: substrate consumption, product formation,
phosphate dissociation and characterisation of a flavin
intermediate. Biochemistry 35, 9907–9916.
19 Hawkes TR, Lewis T, Coggins JR, Mousdale DM,
Lowe DJ & Thorneley RNF (1990) Chorismate
synthase: pre-steady-state kinetics of phosphate release
from 5-enolpyruvylshikimate 3-phosphate. Biochem J
265, 899–902.
20 Bornemann S, Ramjee MK, Balasubramanian S,
Abell C, Coggins JR, Lowe DJ & Thorneley RNF