Crystal Structure of Escherichia coli Enterobactin-specific Isochorismate Synthase (EntC) Bound to its Reaction Product Isochorismate: Implications for the Enzyme Mechanism and Differential Activity of Chorismate-utilizing Enzymes

Article abstract of DOI:10.1016/j.jmb.2010.01.019

EntC, one of two isochorismate synthases in Escherichia coli, is specific to the biosynthesis of the siderophore enterobactin. Here, we report the crystal structure of EntC in complex with isochorismate and Mg²⁺at 2.3 A resolution, the first structure of a chorismate-utilizing enzyme with a non-aromatic reaction product. EntC exhibits a complex α+β fold like the other chorismate-utilizing enzymes, such as salicylate synthase and anthranilate synthase. Comparison of active site structures allowed the identification of several residues, not discussed previously, that might be important for the isochorismate activity of the EntC. Although EntC, MenF and Irp9 all convert chorismate to isochorismate, only Irp9 subsequently exhibits isochorismate pyruvate lyase activity resulting in the formation of salicylate and pyruvate as the reaction products. With a view to understanding the roles of these amino acid residues in the conversion of chorismate to isochorismate and to obtaining clues about the pyruvate lyase activity of Irp9, several mutants of EntC were generated in which the selected residues in EntC were substituted for those of Irp9: these included A303T, L304A, F327Y, I346L and F359Q mutations. Biochemical analysis of these mutants indicated that the side chain of A303 in EntC may be crucial in the orientation of the carbonyl to allow formation of a hydrogen bond with isochorismate. Some mutations, such as L304A and F359Q, give rise to a loss of catalytic activity, whereas others, such as F327Y and I346L, show that subtle changes in the otherwise closely similar active sites influence activity. We did not find a combination of these residues that conferred pyruvate lyase activity.

Full text of DOI:10.1016/j.jmb.2010.01.019

doi:10.1016/j.jmb.2010.01.019
J. Mol. Biol. (2010) 397, 290–300
Available online at www.sciencedirect.com
Crystal Structure of Escherichia coli
Enterobactin-specific Isochorismate Synthase (EntC)
Bound to its Reaction Product Isochorismate:
Implications for the Enzyme Mechanism and Differential
Activity of Chorismate-utilizing Enzymes
1
2
2
Sudharsan Sridharan , Nigel Howard , Olivier Kerbarh ,
1
2
1
Michał Błaszczyk , Chris Abell ⁎ and Tom L. Blundell
1Department of Biochemistry,
University of Cambridge,
EntC, one of two isochorismate synthases in Escherichia coli, is specific to the
biosynthesis of the siderophore enterobactin. Here, we report the crystal
2
+
8
0 Tennis Court Road,
structure of EntC in complex with isochorismate and Mg at 2.3 Å
resolution, the first structure of a chorismate-utilizing enzyme with a non-
aromatic reaction product. EntC exhibits a complex α+β fold like the other
chorismate-utilizing enzymes, such as salicylate synthase and anthranilate
synthase. Comparison of active site structures allowed the identification of
several residues, not discussed previously, that might be important for the
isochorismate activity of the EntC. Although EntC, MenF and Irp9 all
convert chorismate to isochorismate, only Irp9 subsequently exhibits
isochorismate pyruvate lyase activity resulting in the formation of salicylate
and pyruvate as the reaction products. With a view to understanding the
roles of these amino acid residues in the conversion of chorismate to
isochorismate and to obtaining clues about the pyruvate lyase activity of
Irp9, several mutants of EntC were generated in which the selected residues
in EntC were substituted for those of Irp9: these included A303T, L304A,
F327Y, I346L and F359Q mutations. Biochemical analysis of these mutants
indicated that the side chain of A303 in EntC may be crucial in the
orientation of the carbonyl to allow formation of a hydrogen bond with
isochorismate. Some mutations, such as L304A and F359Q, give rise to a loss
of catalytic activity, whereas others, such as F327Y and I346L, show that
subtle changes in the otherwise closely similar active sites influence activity.
We did not find a combination of these residues that conferred pyruvate
lyase activity.
Cambridge CB2 1GA, UK
2Department of Chemistry,
The University Chemical
Laboratory, Lensfield Road,
Cambridge CB2 1EW, UK
Received 20 July 2009;
received in revised form
7
January 2010;
accepted 7 January 2010
Available online
1
5 January 2010
©
2010 Elsevier Ltd. All rights reserved.
Keywords: chorismate-utilizing enzymes; siderophores; enterobactin; EntC;
chorismate isochorismate
Edited by M. Guss
1
from their environment. Chorismate, the product of
Introduction
the shikimate pathway, is successively converted in
2
,3
Escherichia coli by the enzymatic activities of EntC,
Iron-chelating siderophores are essential for the
survival of pathogenic bacteria under the iron-
deficient conditions inside a human host. Enterobac-
tin is the most powerful iron chelator known, with an
estimated association constant of 10
used by some bacteria to compete effectively for iron
4
,5
6
EntB and EntA to 2,3-dihydroxybenzoate, from
5
5
which enterobactin is produced by EntE, EntD and
7
EntF. The genes responsible for the biosynthesis of
5
2
-1
enterobactin are arranged in two bidirectional
clusters. The entCEBA(X) operon forms one cluster
and entD and entF are encoded in the other. The open
reading frame entX, which follows entA in the same
M
and is
8
,9
*
Corresponding author. E-mail address: ca26@cam.ac.uk.
operon is of unknown function.
EntC catalyses the conversion of chorismate to
isochorismate (Fig. 1). The enzyme belongs to a
Present address: S. Sridharan, MedImmune, Aaron
Klug Building, Granta Park, Cambridge, CB21 6GH, UK.
0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.

Crystal Structure of Escherichia coli EntC
291
Fig. 1. Transformations catalysed by chorismate-utilizing enzymes.
2
0
superfamily that utilize chorismate as the substrate
the active site was reported. A tryptophan binding
site has been identified in anthranilate synthase
(TrpE component) and aminodeoxychorismate syn-
1
0
(
Fig. 1). The isochorismate synthase activity of
EntC is specific to the enterobactin biosynthesis
pathway. In addition, E. coli has a menaquinone
biosynthesis pathway-specific isochorismate syn-
2
1
thase (PabB) . Tryptophan is involved in feedback
inhibition of anthranilate synthase but appears to
play only a structural role in PabB.
1
1–13
21
thase activity encoded by the menF gene.
Enterobactin is synthesised under iron-deficient
conditions, whereas menaquinones are required for
electron transport under anaerobic conditions. As a
consequence, the gene clusters responsible for the
synthesis of enterobactin and menaquinones are
regulated differently. Buss et al. also showed that in
E. coli there is channelling of isochorismate in both
these pathways and this may be related to the
relative chromosomal positions of the genes respon-
sible for the biosynthesis of these two compounds in
Chorismate-utilizing enzymes catalyse distinct
transformations of chorismate (Fig. 1). The reactions
all involve initial loss of the C4 hydroxyl group. In
the reaction catalysed by isochorismate synthase
this is replaced by a water molecule that attacks at
C4; however, the precise mechanism for this
1
0
reaction is not known. In anthranilate synthase
(TrpE-TrpG complex) the nucleophile at C2 is
ammonia, which is generated by the glutaminase
(TrpG) subunit and transferred to the bifunctional
1
4
15
their respective gene clusters.
TrpE subunit.
The TrpE subsequently performs
The X-ray crystal structures of several chorismate-
utilizing enzymes are known. Knochel et al. first
reported the structure of Sulfolobus solfataricus
the conversion of chorismate to 2-aminodeoxychor-
ismate, followed by the elimination of pyruvate
(pyruvate lyase activity). Salicylate synthase (Irp9) is
a bifunctional enzyme that initially catalyses the
conversion of chorismate to isochorismate and the
1
5
anthranilate synthase TrpE/TrpG. The structures
of anthranilate synthase homologues from Salmo-
1
6
17
22,23
nella typhimurium and Serratia marcescens were
subsequent formation of salicylate and pyruvate.
subsequently solved. We reported the first structure
In PabB, the side chain of Lys274 acts as the C2
nucleophile to form an enzyme-bound intermediate
that subsequently reacts at C4 with ammonia,
which is generated by an associated glutaminase
1
8
of a salicylate synthase (Yersenia enterocolitica Irp9)
and the structure of its homologue from Mycobacte-
1
9
rium tuberculosis was subsequently described. The
structure of E. coli isochorismate synthase MenF was
2
4,21
(PabA).
1
0
reported by Kolappan et al. and, more recently, a
structure of MenF with Mg and sulfate bound in
Here, we report the first X-ray crystal structure of
2
+
E. coli EntC bound to isochorismate. The roles of

292
Crystal Structure of Escherichia coli EntC
some of the residues in and close to the active site
have been studied using biochemical assays of
specific mutants. The mutated residues were chosen
primarily on the basis of structural comparison with
Irp9. We have identified residues in order to address
the question of why EntC catalyses only the
chorismate to isochorismate conversion, whereas
Irp9 also catalyses the subsequent conversion of
isochorismate to salicylate.
structure of this family and may be regarded as
the representative core enzyme. Although EntC has
only 20–28 % sequence identity with other family
members, they exhibit a high degree of structural
similarity (Supplementary Data Table 1). As can be
seen in the structure-based sequence alignment for
representative proteins (Fig. 3), the most conserved
region is towards the C-terminus.
The active site of EntC
The active site of EntC is located at the base of a
cleft formed by parts of α7, β15, and the loops or
turns between α8 and α9, β18 and β19, β19 and β20,
and β21 and α10 (Fig. 2a). In the active site there is
clear density for isochorismate, the product of the
EntC-catalysed reaction, and a magnesium ion
(Supplementary Data Fig. S3). The ring of the
isochorismate lies approximately between the side
chain of a conserved lysine (K380) on one side and
the carbonyl of the peptide bond between L212 and
A213 on the other (Fig. 2b). The C2 hydroxyl of the
isochorismate is hydrogen bonded to the carbonyl of
A303. The enol-pyruvyl group of the bound
isochorismate is sandwiched between the side
chains of K380 and I346. One of the enol-pyruvyl
carboxylate oxygens is hydrogen bonded to the η1
and ɛ-nitrogens of R347 and the other is hydrogen
bonded to the backbone amide nitrogen of G361 and
a water molecule. This water molecule makes a
hydrogen bond with the backbone amide nitrogen
of R347 and backbone carbonyl of F359 (Fig. 4a). The
magnesium ion is coordinated by the C1 carboxylate
of isochorismate and the side chains of conserved
E241 and E376 residues (Fig. 4a).
Results and Discussion
Structure of EntC
The structure of E. coli EntC (Fig. 2) was solved
using single isomorphous replacement with anom-
alous scattering with wild type and selenomethionyl
derivative datasets. The structure was refined at
2
.3 Å resolution with a final R-factor of 20.0% and an
Rfree of 25.5% (Table 1). There are two protein
molecules, each with an isochorismate molecule
bound in the active site, 293 water molecules, and
four magnesium ions (two per protein molecule) in
the asymmetric unit. The two protein molecules in
the asymmetric unit are related by improper non-
crystallographic symmetry. Clear electron density
was observed for residues 14–391 for both protein
α
chains in the asymmetric unit. The C positions of
the two chains could be superposed with an RMSD
of 0.35 Å.
EntC adopts an α+β fold similar to that of the
known structures of other chorismate-utilizing
1
5
enzymes. The overall structure has nine α-helices
and 18 β-strands. Strands β2, β3, β7 and β11 form a
twisted β-sheet, which makes an orthogonal β-
sandwich with a second twisted sheet formed from
strands β5, β6, β17 and β18. Residues L212, A213,
G214 and S215 of β15 together with H276 of β17 line
the active site. The α-helices lie on the surface of the
molecule.
This is the first structure in which a non-aromatic
product is found bound at the active site of a
chorismate-utilizing enzyme. The positions of the
isochorismate ring and enol-pyruvyl side chain
bound in the active site of EntC are very similar to
those of the salicylate ring and pyruvate, respec-
tively, bound in the active site of salicylate synthase
(Irp9) (Fig. 4c). The interactions of the bound
products are correspondingly similar. The magne-
sium ion is bound in identical positions in EntC,
MenF, and Irp9, with equivalent ligands (Fig. 4;
Supplementary Data Fig. S2). The mutation of the
arginine residue (R387A) in MenF that corresponds
to R347 in the pyruvyl binding pocket in EntC has
Superposition of the structures of the E. coli
menaquinone-specific isochorismate synthase
(
MenF) and EntC demonstrates the existence of
additional secondary structural elements in MenF
formed by residues 1–40 and 118–154. Similarly, the
structures of other chorismate-utilizing enzymes
show additional secondary structural elements in
the N-terminal halves when compared with the
EntC structure. EntC contains a helix, labelled α2a
1
0
been reported to result in inactive enzyme.
There are several residues in EntC thought to be
involved in catalysis that are conserved amongst the
chorismate-utilizing enzymes (Fig. 4a). K147 corre-
sponds to K190 in MenF, which was proposed to act
as the catalytic base by activating a nucleophilic
water molecule, which is also seen hydrogen
bonded to the C2 hydroxyl of isochorismate in the
active site. The carboxyl group of E197 (MenF E240)
points towards C4 of the bound isochorismate, and
is thought to act as the general acid for the loss of the
C4 hydroxyl group from chorismate. This is
consistent with the observation that MenF E240Q
is inactive (Scheme 1).
(
Figs. 2 and 3; and Supplementary Data Fig.S1;
secondary structural elements of EntC labelled
1
5
following the scheme of ssTrpE ), which is not
seen in any of the structures compared here. α2a is
located between β7 and β9, which are common to all
these structures. Its position is almost perpendicular
to that of α1, as seen in all other structures compared
here, except in EntC. β7 continues as β8, which is
absent from EntC. The presence of α2a and its
positioning are both unique to EntC amongst the
chorismate-utilizing enzymes. EntC has the shortest
sequence (391 residues) and the most compact

Crystal Structure of Escherichia coli EntC
293
Fig. 2 (legend on next page)

294
Crystal Structure of Escherichia coli EntC
Table 1
EntC, whereas MenF has tyrosine (Y368 and Y399)
and Irp9 has a tyrosine (Y372) and a glutamine
Native
Se peak
(Q403) at equivalent positions. Interestingly, the
A. Data collection
Space group
orientations of the side chains of F327 and F359 in
EntC are significantly different from the equivalent
residues in MenF and Irp9 (Fig. 4; Supplementary
Data Fig. S2). Among the five enzymes compared in
the structural alignment (Fig. 3; Supplementary
Data Fig. S1) only EntC shows F327 pointing away
P2
1
2
1
2
1
1 1 1
P2 2 2
Resolution (Å)
No. unique reflections
Unit cell dimensions
a (Å)
50.0–2.30
30.0–3.0
41,386
18,988
62.5
104.8
140.0
90.0
62.5
104.5
140.1
90.0
b (Å)
c (Å)
ε1
from the active site with the C atom of the ring
α= β= γ (°)
ζ
approximately occupying the position of C atom of
Matthews coefficient (%)
2.58 (52.3)
2.66
53.8)
(
MenF Y399. In Irp9, the equivalent tyrosine (Y372)
points towards the active site with its side chain
hydroxyl oxygen at a distance of 2.5 Å from O1 of
the bound pyruvate. The equivalent tyrosine resi-
dues in MenF and TrpE and the tryptophan in PabB
point towards the active site in these enzymes. The
residue at position 359 is not conserved in the five
chorismate-utilizing enzymes compared here. F359
of EntC points towards the active site and its ring
partially occupies the position of Y372 in Irp9. The
equivalent tyrosine (Y399) in MenF points away
from the active site in the direction of and partially
occupying the position of the EntC F327 ring. The
implications of these differences are discussed later.
EntC, MenF and Irp9 all convert chorismate to
isochorismate; however, only Irp9 subsequently
exhibits isochorismate pyruvate lyase activity result-
ing in the formation of salicylate and pyruvate as the
reaction products. Several active site residues of
chorismate-utilizing enzymes have been studied
High resolution shell (Å)
I/σI overall
Completeness overall
Redundancy overall
2.36–2.30
3.11–3.0
12.4 (1.6) 16.3 (4.6)
99.9 (99.8) 100 (100)
6.4 (5.1)
14.7
14.9)
(
Phasing power
ISO (acentric/centric)
ANO
0.36/0.30
0.82
—
—
—
FOM (acentric/centric)
0.133/
0
.085
B. Refinement statistics
Resolution (Å)
50.0-2.30
2088
39292
2
5856
297
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
No. reflections in test set
No. reflections in working set
No. protein molecules in asymmetric unit
No. protein atoms in asymmetric unit
No. water molecules in asymmetric unit
R-factor (%)
20.0
25.5
Rfree (%)
RMSD from ideal
Bond lengths (Å)
Bond angles (°)
0.01
1.4
2
Average B-factors (Å )
Main chain
Side chain and water molecules
RMS B main chain
RMS B side chain
using mutation analysis to understand the roles of
46.0
48.6
1.9
16,21
these residues in catalysis of these enzymes.
We
have attempted single, double, triple and quadruple
amino acid substitutions in EntC (Table 2) at the
non-conserved positions 303, 304, 327, 346 and 359,
in an attempt to shed light on the roles of these
residues in EntC activity and to gain an understand-
ing of the differences in activity compared to Irp9.
We chose to substitute the residues at these positions
in EntC with the equivalent residues from Irp9 (i.e.
A303T, L304A, F327Y, I346L and F359Q) in order to
see if any of these substitutions singly or in
combination with others could impart salicylate
synthase activity to EntC.
3.8
Values within parentheses are for the highest resolution shell.
Studies on wild type and mutant forms of EntC
Structural alignment and comparison of the active
sites of EntC, MenF and Irp9 reveals a high degree of
conservation and points of differences. The residues
corresponding to K147, E197, L212-S215, E241,
H276, R347, A360-A362, E376 and K380 are con-
served among these enzymes (Figs. 3 and 4). H276,
L212-S215 and A360-A362 interact directly with the
product in EntC. The nearby residues A303, L304
and I346 are not strictly conserved in the three
enzymes. A303 is conserved between EntC and
MenF but replaced by threonine in Irp9. L304 is
substituted by valine in MenF and alanine in Irp9.
I346 is replaced by leucine in both MenF and Irp9.
Structural comparison also reveals that the residues
near the enol-pyruvyl carboxylate of the bound
isochorismate are not conserved in these enzymes.
There is phenylalanine at positions 327 and 359 in
The various mutant EntC enzymes containing
double, triple and quadruple amino acid substitu-
tions (Table 2) either have significantly reduced
isochorismate synthase activity or are inactive. As
described in Materials and Methods, salicylate
production was monitored in the absence of PchB
(which converts isochorismate to salicylate). In these
assays, neither wild type EntC nor any of the mutant
enzymes showed any salicylate synthase activity.
Residues A303, L304, I346 are aligned over C3 and
the enolpyruvate group of isochorismate (Fig. 4a),
with the backbone carbonyl oxygen of A303 only
Fig. 2. (a) The structure of E. coli EntC shown in cartoon representation using rainbow colours from the N to the C
terminus. The bubble encompasses the active site region of EntC, shown enlarged in (b). Isochorismate, grey; magnesium
ion, green sphere; and water, blue sphere. Several active site residues are shown but, for clarity, only the side chains of the
residues are shown. The figures were generated using PyMOL (www.pymol.org).

Crystal Structure of Escherichia coli EntC
295
Fig. 3. Structure-based sequence alignment of representative chorismate-utilizing enzymes and EntC sequences
3
4
generated using the ESPript program. ecEntC, E. coli enterobactin-specific isochorismate synthase; ecMenF, E. coli
menaquinone-specific isochorismate synthase; yeIrp9, Y. enterocolitica salicylate synthase; ssTrpE, S. sulfotoricus
anthranilate synthase TrpE component; ecPabB, E. coli p-aminodeoxyisochorismate synthase. The highly variable N-
terminal sequence is not shown in the alignment. The numbering scheme used for the secondary structure elements of
1
5
ecEntC follows that of ssTrpE. α2a is unique for ecEntC, which also lacks α1.
3
.1 Å from the C2 hydroxyl oxygen. Mutations in
(chorismate) for the A303T EntC mutant was found
to be 130-fold lower than that for wild type EntC.
Further, all double, triple and quadruple amino acid
substitutions made involving this A303T mutation
resulted in an enzyme with no detectable activity.
The analogous mutation in MenF (A344T) has been
this region therefore would be expected to affect
activity. The I346L EntC mutant has a 12-fold lower
kcat/K (chorismate) than the wild type enzyme and
m
the L304A, F359Q double mutation resulted in an
enzyme with no detectable activity. The kcat/Km

296
Crystal Structure of Escherichia coli EntC
Fig. 4. Active sites of EntC (a), MenF (b) and Irp9 (c). The expected magnesium ion is shown as a green sphere and
water molecules are shown as blue spheres. ISC, isochorismate; SAL, salicylate; PYR, pyruvate. For clarity, only some of
the hydrogen bonding interactions are shown (dotted lines).

Crystal Structure of Escherichia coli EntC
297
Scheme 1. Proposed mechanism of EntC reaction.
reported to result in complete loss of activity and
consequently a role was proposed for this residue in
which is calculated to be 8.0 – 10.1 kcal/mol less
stable than the lowest energy pseudo-equatorial
1
0
25
the isomerisation step. For nucleophilic attack at
the C2 position of chorismate to give the product
with the correct geometry, the water molecule needs
to approach the chorismate from the si-face, The
EntC structure shows that there is an interaction
between the backbone carbonyl group of A303 and
isochorismate, which suggests a similar interaction
between the backbone carbonyl and the water
involved in the nucleophilic attack at C4 of
chorismate. Changing the size of the side chain of
this residue presumably results in some reorienta-
tion of the backbone carbonyl, which could well
perturb the transition state. The other residue
contributing to the si-face of water at the C2 position
is K147, the lysine being required to activate the
water for nucleophilic attack.
The decrease in the activity of EntC incorporating
L304A and I346L substitutions shows that subtle
changes in the active site affect the ability of the
enzyme to hold the substrate in an appropriate
conformation for the reaction to proceed. The crystal
structure of EntC with the product bound shows that
it binds isochorismate in a pseudo-axial conformation,
conformation. It is postulated that the [3,3] sigma-
tropic rearrangements from chorismate to prephe-
2
6
nate and isochorismate to isoprephenate and the
[1,5] sigmatropic re-arrangement from isochorismate
to salicylate and pyruvate (Scheme 2) requires
adoption of the higher energy diaxial conformation.
The active site cavity in EntC is slightly larger than
that in Irp9, and possesses a small hydrophobic
pocket formed by residues Leu304 and Gly361,
which is absent from Irp9. The enol-pyruvyl group
binds towards this pocket in such a way that the
resultant dihedral angle for C2-C3-O-C8 is 72° with
the side chain held away from the central ring. This
conformation is unlikely to facilitate the sigmatropic
re-arrangements, and could explain both the lack of
pyruvate lyase activity of EntC ([1,5] sigmatropic re-
arrangement) and the absence of side-products
associated with [3,3] sigmatropic rearrangements of
either substrate (chorismate) and product (isochor-
3
ismate) observation.
The differences in orientation of the side chains of
F327 and F359 in EntC compared with other
chorismate-utilizing enzymes (Fig. 4) prompted
investigation of the effects of amino acid substitutions
in this region. The F327Y and F359Q mutations
resulted respectively in 48-fold and 10-fold lower
kcat/Km than the wild type EntC enzyme. Surpris-
ingly, the F327Y/F359Q double mutation showed
Table 2. Isochorismate synthase activity assay for wild-
type and mutant EntC
K
m
kcat/K
m
only an eightfold reduction in kcat/K . It is not clear
m
k
cat
(min )
(chorismate) (chorismate)
-
1
-1 -1
whether the substitution at position 359 can partially
rescue the loss of activity resulting from substitution
at position 327. Enzymes with double mutations
F327Y/I346L or F359Q/I346L have 750-fold and 130-
fold lower kcat/Km than the wild type enzyme.
Comparison of the architectures of EntC, MenF and
Irp9 in this region shows that it may be possible for
these residues in EntC to be positioned in orientations
similar to that in MenF. However, the EntC structure
shows that tyrosine at position 327 can be accommo-
dated in the same orientation as the phenylalanine in
the wild type enzyme. It has not been possible to
reconcile the results of activity assays with structural
information as the enzymes incorporating the F327Y
and F359Q substitutions could not be crystallised.
Studies on the Irp9 Y327F mutant (corresponding to
Mutation
(μM)
(min μM )
Wild type EntC
A303T
37
4
7 ± 0.8
103 ± 17
76 ± 13
76 ± 8
66 ± 10
261 ± 62
466 ± 85
40 ± 9
—
5.3
0.04
0.1
0.4
0.5
0.04
0.006
0.65
—
—
—
—
—
F327Y
8
I346L
F359Q
F359Q,I346L
F327Y,I346L
F327Y,F359Q
A303T,F327Y
A303T,F359Q
L304A,F359Q
A303T,F327Y, F359Q
A303T,F327Y,I346L
F327Y,F359Q,I346L
A303T,F327Y,F359Q,I346L
33
36
11
3
26
na
na
na
na
na
na
na
—
—
—
—
—
—
—
—
na, no detectable activity under experimental conditions.

298
Crystal Structure of Escherichia coli EntC
Scheme 2. Sigmatropic re-arrangements of chorismate and isochorismate.
F327 in EntC) confirmed that the tyrosine is not
involved in a protonation mechanism for pyruvate
cooled to 22 °C. IPTG was added to a final concentration of
1
mM to induce EntC over-expression in the cells and the
1
8
medium was incubated for 12–14 h at the same temperature
with shaking at 220 rpm.
elimination in this enzyme.
EntC and related chorismate-utilizing enzymes
are involved in essential biosynthetic pathways in
pathogenic bacteria. In this context, the availability
of the structure of EntC with product bound is
important for providing insight into the enzyme
mechanism and for the rational design of small
molecule inhibitors. Our studies show that although
the chorismate-utilizing enzymes all have highly
similar active site architectures, subtle differences in
the active site residues are enough for these enzymes
to acquire a distinct biochemical activity. However,
mutagenesis studies have proved to be too blunt a
tool to provide a clear picture of precisely how the
catalytic activities are differentiated.
The cells were harvested by centrifugation at 3000 g for
0 min at 4 °C and the pelleted cells were resuspended in
3
buffer A (20 mM Tris pH 7.5, 0.5 M NaCl, 2 mM β-
mercaptoethanol) at 4 °C. DNase I at a final concentration
of 10 μg/ml and Protease Inhibitor Cocktail (Calbiochem)
were added to the suspension before cell lysis using
sonication at 4 °C. The crude lysate was clarified by
centrifugation at 10,000g for 60 min maintaining the
temperature at 4 °C. The clarified lysate was filtered
(
0.22 μm pore size) before subsequent chromatographic
purification steps at 4 °C.
EntC was purified to near-homogeneity using nickel
affinity and gel-filtration chromatography, with perfor-
mance being monitored by SDS–PAGE. The clarified
2+
lysate was passed through the Ni -charged HiTrap
chelating agarose columns (GE Life Sciences) equilibrated
with buffer A. The columns were washed with five
column volumes of buffer A to remove contaminant
proteins. EntC bound to the matrix in the columns was
eluted using increasing concentrations of imidazole using
buffer B (20 mM Tris pH 7.5, 0.5 M NaCl, 0.5 mM
imidazole). The bound protein was found to elute at
150 mM and higher concentrations of imidazole. The
eluted protein was concentrated and the buffer changed
to buffer C (20 mM Tris pH 7.5, 5 mM magnesium
chloride, 1 mM DTT) using a PD-10 desalting column
packed with Sephadex G-25 resin (GE Life Sciences). The
protein was concentrated and further purified using a
XK-16/60 Superdex 75 size-exclusion column (GE Life
Sciences) after equilibration with buffer C. The hexahis-
tidine tag of the purified protein was cleaved using
thrombin (Novagen) in an overnight reaction performed
at 4 °C in a total volume of 50 ml. The sample was
concentrated and the size-exclusion chromatographic
step described above was done again to remove thrombin
and the cleaved hexahistidine tag from EntC. The clones
for the various EntC mutant proteins were generated
Materials and Methods
Cloning, over-expression and purification of EntC
The entC gene (1176 bp) from E. coli K12 was cloned using
the upstream primer: 5′-CATATGGATACGT-
CACTGGCT-3′ with an NdeI restriction site (bold letters)
and the downstream primer: 5′-CTCGAGTCATTAATG-
CAATCC-3′ with an XhoI restriction site (bold letters). The
amplified product was then digested with the restriction
enzymes and ligated with similarly cut pET-28a plasmid
DNA (Novagen). The identity of the construct was verified
by nucleotide sequencing. For over-expression of EntC, C41
(
DE3) or BL21 (DE3) cells (Stratagene) were transformed
with the above plasmid containing the entC gene. Trans-
formants were selected by growth at 37 °C on Luria-Bertani
(
LB) agar medium containing kanamycin (50 μg/ml). A
single colony was picked and used to inoculate 100 ml of
2
×YT medium containing kanamycin (50 μg/ml). This
starter culture was used to inoculate 4 l of 2×YT medium.
After this culture reached an A600nm of 0.6–0.8 unit, it was

Crystal Structure of Escherichia coli EntC
299
using the QuikChange site-directed mutagenesis protocol
Acknowledgements
(
Stratagene). These mutants were over-expressed and
purified using the protocol used for wild type protein as
described above. The selenomethionyl derivative of EntC
was over-expressed in BL21(DE3) cells using metabolic
We thank Dr Dima Chirgadze at the Department
of Biochemistry, University of Cambridge for help
with X-ray data collection and discussion of the
EntC structure. We thank the BBRSC for support of
this project (RG43711).
2
7
inhibition procedure.
Enzymatic assays
Salicylate formation by wild type and mutant EntC
enzymes was monitored by measuring fluorescence
Supplementary Data
(
excitation at 300 nm, excitation at 420 nm). The reactions
contained 200–500 nM of wild type or mutant EntC in
00 mM phosphate pH 7.0, 10 mM MgCl . Isochorismate
Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.jmb.2010.01.019
1
2
synthase activity of wild type or mutant EntC enzymes
was measured using a coupled enzyme assay with
Pseudomonas aeruginosa isochorismate pyruvate lyase
(
PchB) as the second enzyme, which converts isochor-
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