Biochemical characterization and inhibitor discovery
of shikimate dehydrogenase from Helicobacter pylori
Cong Han
1
, Lirui Wang
1
, Kunqian Yu
1
, Lili Chen
1
, Lihong Hu
1
, Kaixian Chen
1
, Hualiang Jiang
1,2
and Xu Shen
1,2
1 Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai, China
2 School of Pharmacy, East China University of Science and Technology, Shanghai, China
Helicobacter pylori is a gram-negative, microaerophilic,
motile, and spiral-shaped bacterium that colonizes the
gastric mucosa. Since it was discovered by Marshall
and Warren in 1982 [1], H. pylori has been recognized
as one of the most common human pathogens, prob-
ably infecting about 50% of the world’s human popu-
lation [2]. H. pylori is a major causative factor for
several gastrointestinal illnesses, including gastritis,
Keywords
antibacterial agent; drug target; enzyme

and K
m
of 0.148 mm
toward shikimate, k
cat
of 7.1 s
)1
and K
m
of 0.182 mm toward NADP, k
cat
of 5.2 s
)1
and K
m
of 2.9 mm toward NAD. The optimum pH of the
enzyme activity is between 8.0 and 9.0, and the optimum temperature is
around 60 °C. Using high throughput screening against our laboratory
chemical library, five compounds, curcumin (1), 3-(2-naphthyloxy)-4-oxo-
2-(trifluoromethyl)-4H-chromen-7-yl 3-chlorobenzoate (2), butyl 2-{[3-
(2-naphthyloxy)-4-oxo-2-(trifluoromethyl)-4H-chromen-7-yl]oxy}propanoate
(3), 2-({2-[(2-{[2-(2,3-dimethylanilino)-2-oxoethyl]sulfanyl}-1,3-benzothiazol-
6-yl)amino]-2-oxoethyl}sulfanyl)-N-(2-naphthyl)acetamide (4), and maes-
aquinone diacetate (5) were discovered as HpSDH inhibitors with IC
50
values of 15.4, 3.9, 13.4, 2.9, and 3.5 lm, respectively. Further investigation
indicates that compounds 1, 2, 3, and 5 demonstrate noncompetitive inhibi-
tion pattern, and compound 4 displays competitive inhibition pattern with
respect to shikimate. Compounds 1, 4, and 5 display noncompetitive inhibi-
tion mode, and compounds 2 and 3 show competitive inhibition mode with

pathogenic bacterium [6,7]. The development of bacter-
ial genomics has provided investigators with powerful
tools to identify novel antibacterial targets [8,9]. At the
same time, comparison of bacterial target genes with
human genes will also be necessary because, to avoid
adverse effects, a good antimicrobial drug target
should have no homolog in mammalian cells.
In bacteria, erythrose 4-phosphate is converted to
chorismate through seven steps in the shikimate path-
way, which is essential for the synthesis of important
metabolites, such as aromatic amino acids, folic acid,
and ubiquinone [10]. The shikimate pathway is crucial
to algae, higher plants, bacteria and fungi, but absent
in mammals [11,12]. Therefore, the enzymes involved
in this pathway have received much attention as poten-
tial drug targets for developing nontoxic antimicrobial
agents, herbicides, and antiparasite drugs [13]. For
example, the compound glyphosate produced by
Monsanto Company was proved to be one of the
world’s best-selling herbicides. It has been determined
as the inhibitor of 5-enoylpyruvyl shikimate phosphate
synthase (EPSP synthase) and has shown potent inhib-
itory activity against the growth of apicomplexan para-
sites in vitro [12]. The compound 6(S)-fluoroshikimate,
produced by AstraZeneca Inc. (London, UK), is con-
verted to 6-fluorochorismate by the subsequent
enzymes in the shikimate pathway, thus 6(S)-fluoros-
hikimate could block the biosynthesis of p-aminoben-
zoic acid and inhibit the growth of Escherichia coli
[14,15]. In addition, a number of enzyme inhibitors

substrate and NADP cofactor. The detailed structural
information might expedite the discovery of novel
SDH inhibitors and further of antimicrobial agents,
though few SDH inhibitors have yet been reported so
far.
In this work, we identified a new aroE gene enco-
ding SDH from H. pylori strain SS1. The recombinant
H. pylori shikimate dehydrogenase (HpSDH) was
cloned, expressed, and purified in E. coli system, and
its biochemical and enzymatic characterizations were
also carried out. Furthermore, by using the high-
throughput screening technology, five novel HpSDH
inhibitors were discovered and their antibacterial activ-
ities were also assayed. This study is expected to help
better understand the features of SDH and provide
useful information for the development of novel anti-
biotics to treat H. pylori-associated infection.
Results and Discussion
Cloning, expression, and sequence analysis of
HpSDH
In the current work, the aroE gene of H. pylori strain
SS1 was cloned by using the genome sequences of
H. pylori strains 26695 and J99 as major references.
We firstly amplified a DNA fragment including the
entire coding region of HpSDH in order to identify
the exact aroE gene sequence. On the basis of the
C. Han et al. H. pylori shikimate dehydrogenase
FEBS Journal 273 (2006) 4682–4692 ª 2006 The Authors Journal compilation ª 2006 FEBS 4683
sequencing result from PCR products, we synthesized
two oligonucleotides for cloning the aroE gene. The

4684 FEBS Journal 273 (2006) 4682–4692 ª 2006 The Authors Journal compilation ª 2006 FEBS
alanine residues of the conserved sequence motif
GAGGA are replaced by two serine residues in
H. pylori. Thus, the binding interaction of NADP to
HpSDH might be different from those of NADP to
the other SDHs. HpSDH is around 31, 31, 33, 30, 30,
and 26% identical to E. coli, H. influenzae, Neisseria
meningitidis, M. jannaschii, Archaeoglobus fulgidus, and
Mycobacterium tuberculosis SDH, respectively.
To obtain the high level of protein production, we
reduced the amount of isopropyl thio-b-d-galactoside
(IPTG) and culture temperature to avoid the possible
formation of inclusion body in the expression
approach. After one-step purification of nickel-affinity
chromatography, the recombinant HpSDH, coupled
with a C-terminus six-histidine tag, was purified to
apparent homogeneity (Fig. 2).
Characterization of the recombinant HpSDH
The LC ⁄ MS spectral data (Fig. 3) give a 30 038 Da
molecular mass of the recombinant HpSDH, which is
in good agreement with the theoretical molecular
mass of 30 041 Da calculated according to the amino
acid sequence. This result thereby demonstrates
the veracity of the expressed recombinant HpSDH.
The circular dichroism (CD) spectrum reveals that
the percentages for a-helix, b-sheet, b-turn, and ran-
dom coil in HpSDH are, respectively, 16.6, 49.2, 1.5,
and 32.6% processed by jasco secondary structure
estimation software. The percentage for random coil
of HpSDH is similar to that (32%) calculated

cat
of 7.1 ± 0.7 s
)1
, K
m
of 0.182 ± 0.027 mm and
k
cat
⁄ K
m
of 3.9 · 10
4
m
)1
Æs
)1
toward NADP. Different
from AroE of E. coli [23], HpSDH can oxidize shiki-
mate using NAD as cofactor, which has a k
cat
of
5.2 ± 0.1 s
)1
and K
m
of 2.9 ± 0.4 mm toward NAD.
HpSDH shows a 10 times higher K
m
for NAD than
for NADP at saturation of shikimate, suggesting that

of HpSDH. It is found that the pH optimum of
HpSDH is between 8.0 and 9.0, and the pH optimum
of AfSDH is between 7 and 7.5 [27]. Both AfSDH and
HpSDH exhibit very low activities at extremely aci-
dic ⁄ basic pH values, while MtSDH still displays higher
enzyme activity at pH 10–12 [26]. It is thus suggested
that the active site of SDH might involve several aci-
dic ⁄ basic amino acid residues that play crucial roles in
the catalytic process.
HpSDH inhibitor discovery
Using high throughput screening against our construc-
ted chemical library containing 5000 compounds, five
compounds, curcumin (1), 3-(2-naphthyloxy)-4-oxo-
2-(trifluoromethyl)-4H-chromen-7-yl 3-chlorobenzoate
(2), butyl 2-{[3-(2-naphthyloxy)-4-oxo-2-(trifluorometh-
yl)-4H-chromen-7-yl]oxy}propanoate (3), 2-({2-[(2-
{[2-(2,3-dimethylanilino)-2-oxoethyl]sulfanyl}-1,3-benzo-
thiazol-6-yl)amino]-2-oxoethyl}sulfanyl)-N-(2-naphthyl)
acetamide (4) and maesaquinone diacetate (5) were
Table 1. Comparison of kinetic parameters of SDH enzymes from
various bacteria.
a
Kinetic parameters for M. tuberculosis SDH are
from [26].
b
Kinetic parameters for E. coli SDH are from [23].
c
Kin-
etic parameters for A. fulgidus SDH are from [27].
SDH

)1
s
)1
)
(NADP)
HpSDH 7.7 0.148 0.182 5.2 · 10
4
3.9 · 10
4
MtSDH
a
399 0.03 0.063 1.33 · 10
7
6.33 · 10
6
EcSDH
b
237 0.065 0.056 3.65 · 10
6
4.23 · 10
6
AfSDH
c
361 0.17 0.19 2.12 · 10
6
1.9 · 10
6
Fig. 4. Temperature profile of HpSDH enzyme activity.
Fig. 5. pH profile of HpSDH enzyme activity.
H. pylori shikimate dehydrogenase C. Han et al.

½S
½Sð1 þ
½I
aK
i
ÞþK
m
ð1 þ
½I
K
i
Þ
ð1Þ
m ¼
V
max
½S
½SþK
m
1 þ
½I
K
i

ð2Þ
Evaluation of antibacterial activity
The determined HpSDH inhibitors were tested for
antibacterial activity against H. pylori. The results
show that compounds 1, 2, and 5 display moderate
inhibitory activity against the growth of H. pylori

C. Han et al. H. pylori shikimate dehydrogenase
FEBS Journal 273 (2006) 4682–4692 ª 2006 The Authors Journal compilation ª 2006 FEBS 4687
Fig. 8. Inhibition of HpSDH toward shikimate by increasing concentrations of compounds 1–5. (A) Compound 1 [0 lM (n), 5 lM (d), 10 lM
(m), and 20 lM (.)]. (B) Compound 2 [0 lM (n), 2.5 lM (d), 5 lM (m), and 10 lM (.)]. (C) Compound 3 [0 lM (n), 5 lM (d), 10 lM (m), and
20 l
M (.)]. (D) Compound 4 [0 lM (n), 1 lM (d), 2.5 lM (m), and 5 lM (.)]. (E) Compound 5 [0 lM (n), 1 lM (d), 5 lM (m), and 10 lM (.)].
Fig. 9. Inhibition of HpSDH toward NADP by increasing concentrations of compounds 1–5. (A) Compound 1 [0 lM (n), 2.5 lM (d), 5 lM (m),
and 10 l
M (.)]. (B) Compound 2 [0 lM (n), 2.5 lM (d), 5 lM (m), and 10 lM (.)]. (C) Compound 3 [0 lM (n), 5 lM (d), 10 lM (m), and 20 lM
(.)]. (D) Compound 4 [0 lM (n), 1 lM (d), 2.5 lM (m), and 5 lM (.)]. (E) Compound 5 [0 lM (n), 2.5 lM (d), 5 lM (m), and 10 lM (.)].
H. pylori shikimate dehydrogenase C. Han et al.
4688 FEBS Journal 273 (2006) 4682–4692 ª 2006 The Authors Journal compilation ª 2006 FEBS
In conclusion, we have firstly cloned and expressed
HpSDH enzyme, and the biochemical characterization
of HpSDH is expected to favor better understanding
the SDH features. Moreover, by high throughput
screening methodology, we have identified and charac-
terized five novel HpSDH inhibitors, and three of
which show moderate inhibition activities against the
growth of H. pylori in vitro. These inhibitors represent
new chemical scaffolds available for further chem-
ical modification in the development of novel SDH
inhibitors with increased specificity and antibacterial
activity.
Experimental procedures
Materials
H. pylori strains SS1 and ATCC 43504 were obtained from
Shanghai Institute of Digestive Disease (Shanghai, China).
E. coli host strain BL21(DE3) was purchased from Strata-
gene (La Jolla, CA, USA). The chemical library containing

Expression and purification of HpSDH
The recombinant clone pET22b-HpSDH was transformed
into E. coli strain BL21(DE3) grown in LB media supple-
mented with 100 lgÆmL
)1
ampicillin at 37 °C. When the
A
600
reached 0.6, the culture was induced by 0.4 mm
IPTG and incubated at 25 °C for additional 6 h. The
cells were harvested by centrifugation and suspended in
buffer A (20 mm Tris ⁄ HCl, pH 8.0, 500 mm NaCl,
10 mm imidazole). After sonication treatment on ice, the
mixture was centrifuged to yield a clear supernatant,
which was loaded onto a column with Ni-NTA resin
(Qiagen, Hilden, Germany) pre-equilibrated in buffer A.
The column was washed with buffer B (20 mm Tris ⁄ HCl,
pH 8.0, 500 mm NaCl, 20 mm imidazole) several times
and eluted with buffer C (20 mm Tris ⁄ HCl, pH 8.0,
500 mm NaCl, 200 mm imidazole), then the eluted frac-
tions were pooled and dialyzed against buffer D (20 mm
Tris ⁄ HCl, pH 8.0, 200 mm NaCl, 5 mm DTT) to remove
imidazole. Fractions containing HpSDH were pooled and
concentrated by ultrafiltration with an Amicon centrifugal
filter device. All purification, dialysis and concentration
procedures were performed at 4 °C. Protein concentration
was determined by Bradford assay using bovine serum
albumin as standard.
Enzymatic activity assay
The enzymatic activity of HpSDH was assayed at 25 °Cby

volume 200 lL, path length 0.6 cm) contained 100 mm
Tris ⁄ HCl (pH 8.0), shikimate and NADP (or NAD) at
desired concentrations. The K
m
and V
max
values for sub-
strates were determined by varying the concentrations of
one substrate while keeping the other substrate at satura-
tion. In the experiment where shikimate was the varied
substrate (0.0625, 0.125, 0.25, 0.5, and 1 mm), the concentr-
ation of NADP was maintained at 2 mm, whereas the
concentration of shikimate was fixed at 2 mm when NADP
was the varied substrate (0.0625, 0.125, 0.25, 0.5, and
1mm). The assay reaction was initiated by the addition
of the diluted HpSDH enzyme. To measure the kinetic
parameters for NAD, the concentration of shikimate was
fixed at 2 mm when NAD was the varied substrate (0.25,
0.5, 1, 2, and 4 mm). The kinetic parameters K
m
and V
max
were calculated from the slope and intercept values of the
linear fit in a Lineweaver–Burk plot. To test the enzymatic
activity of HpSDH in the presence of quinate, the assay
solution consisted of 100 mm Tris ⁄ HCl (pH 8.0), 4 mm qui-
nate, and 2 mm NADP (or NAD). Each measure was taken
in triplicate.
The effects of pH and temperature on HpSDH enzymatic
activity were determined by the above assay method. All

of HpSDH was repeated three times.
Inhibitor discovery
Our chemical library containing 5000 compounds was used
for HpSDH inhibitor screening. Based on the procedure of
enzyme activity assay, the initial velocities of the enzyme
activity were determined in the presence of compounds
(10 lm) dissolved in dimethyl sulfoxide. The final dimethyl
sulfoxide concentration in all assay mixtures was 0.1%
(v ⁄ v). The assay buffer contained 100 mm Tris ⁄ HCl
(pH 8.0), 2 mm shikimate, and 2 mm NADP. The reaction
was initiated by the addition of the diluted HpSDH enzyme
(18 nm). After the preliminary screening, compounds 1–5
were identified to inhibit HpSDH enzyme activity. The ini-
tial velocities of the enzyme activity were determined in the
presence of various concentrations of compounds 1–5
(0–50 lm) to investigate the dose-dependent inhibition
effects. IC
50
values of compounds 1–5 were obtained by fit-
ting the data to a sigmoid dose–response equation of the
origin software (OriginLab, Northampton, MA, USA).
Afterwards, inhibitor modality was determined by measur-
ing the effects of inhibitor concentrations on the enzymatic
activity as a function of substrate concentration. In the
inhibition experiment where the NADP concentration was
fixed at 2 mm, shikimate was a varied substrate (0.0625,
0.125, 0.25, 0.5, and 1 mm) when the concentration of
inhibitor was varied from 0 to 20 lm. In parallel, in the
inhibition experiment where the shikimate concentration
was fixed at 2 mm, NADP was a varied substrate (0.0625,

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