Tài liệu Báo cáo khoa học: Biochemical characterization and inhibitor discovery of shikimate dehydrogenase from Helicobacter pylori docx

Biochemical characterization and inhibitor discovery
of shikimate dehydrogenase from Helicobacter pylori
Cong Han1, Lirui Wang1, Kunqian Yu1, Lili Chen1, Lihong Hu1, Kaixian Chen1, Hualiang Jiang1,2
and Xu Shen1,2
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

Keywords
antibacterial agent; drug target; enzyme
inhibition; Helicobacter pylori; shikimate
dehydrogenase
Correspondence
X. Shen, H. Jiang, and L. Hu, Shanghai
Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zu Chong Zhi
Road, Zhangjiang Hi-Tech Park, Shanghai
201203, China.
Tel ⁄ Fax: +86 21 50806918
E-mail: ,
,

Database
The sequence reported in this paper has
been submitted to GenBank database under
accession number AY738333
(Received 23 April 2006, revised 11 July
2006, accepted 16 August 2006)
doi:10.1111/j.1742-4658.2006.05469.x

Shikimate dehydrogenase (SDH) is the fourth enzyme involved in the shikimate pathway. It catalyzes the NADPH-dependent reduction of 3-dehydroshikimate to shikimate, and has been developed as a promising target

for the discovery of antimicrobial agent. In this report, we identified a new
aroE gene encoding SDH from Helicobacter pylori strain SS1. The recombinant H. pylori shikimate dehydrogenase (HpSDH) was cloned, expressed,
and purified in Escherichia coli system. The enzymatic characterization of
HpSDH demonstrates its activity with kcat of 7.7 s)1 and Km of 0.148 mm
toward shikimate, kcat of 7.1 s)1 and Km of 0.182 mm toward NADP, kcat
of 5.2 s)1 and Km 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-oxo2-(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-benzothiazol6-yl)amino]-2-oxoethyl}sulfanyl)-N-(2-naphthyl)acetamide (4), and maesaquinone diacetate (5) were discovered as HpSDH inhibitors with IC50
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 inhibition pattern, and compound 4 displays competitive inhibition pattern with
respect to shikimate. Compounds 1, 4, and 5 display noncompetitive inhibition mode, and compounds 2 and 3 show competitive inhibition mode with
respect to NADP. Antibacterial assays demonstrate that compounds 1, 2,
and 5 can inhibit the growth of H. pylori with MIC of 16, 16, and
32 lgỈmL)1, respectively. This current work is expected to favor better
understanding the features of SDH and provide useful information for the
development of novel antibiotics to treat H. pylori-associated infection.

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, probably infecting about 50% of the world’s human population [2]. H. pylori is a major causative factor for
several gastrointestinal illnesses, including gastritis,

Abbreviations
AfSDH, Archaeoglobus fulgidus shikimate dehydrogenase; EcSDH, Escherichia coli shikimate dehydrogenase; EPSP synthase, 5-enoylpyruvyl
shikimate phosphate synthase; HpSDH, Helicobacter pylori shikimate dehydrogenase; IPTG, isopropyl thio-b-D-galactoside; MIC, minimal

inhibitory concentration; MtSDH, Mycobacterium tuberculosis shikimate dehydrogenase; SDH, shikimate dehydrogenase.

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C. Han et al.

peptic ulceration, and gastric cancer [3]. It has been
confirmed that the rapid infection of H. pylori is a
severe threat to human health. Currently, combination
therapies employing one proton pump inhibitor (e.g.
omeprazole) and two or three antibiotics (e.g. metronidazole, amoxicillin, and clarithromycin) have been
used as preferred treatment against H. pylori infection
[4]. However, such multiple therapy regiments have
not been very effective in a clinical setting, because the
overuse and misuse of antibacterial agents have resulted in the emergence of antibiotic-resistant strains [5].
Therefore, the alarming rise of antibiotics resistance
among key bacterial pathogens is stimulating an urgent
need to discover novel antibacterial agents acting on
new drug targets. Fortunately, the accomplishment of
H. pylori genome-sequencing project has heralded a
new era for antibacterial chemotherapy against the
pathogenic bacterium [6,7]. The development of bacterial 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 pathway, 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 potential 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 inhibitory activity against the growth of apicomplexan parasites in vitro [12]. The compound 6(S)-fluoroshikimate,
produced by AstraZeneca Inc. (London, UK), is converted to 6-fluorochorismate by the subsequent
enzymes in the shikimate pathway, thus 6(S)-fluoroshikimate could block the biosynthesis of p-aminobenzoic acid and inhibit the growth of Escherichia coli
[14,15]. In addition, a number of enzyme inhibitors
have been prepared to investigate the mechanism of
the enzymes within the shikimate pathway [16,17].
Shikimate dehydrogenase (SDH, EC 1.1.1.25) catalyzes the fourth reaction in the shikimate pathway,
and is responsible for the NADPH-dependent reduction of 3-dehydroshikimate to shikimate. SDH belongs

H. pylori shikimate dehydrogenase

to the superfamily of NAD(P)H-dependent oxidoreductase. In plants, including Pisum sativum and Nicotiana tabacum, SDH is associated with 3-dehydroquinate
dehydratase to form bifunctional enzyme [18,19]. In
fungi and yeast, such as Aspergillus nidulans and Saccharomyces cerevisiae, SDH exists as a component of
the penta-functional AROM enzyme complex that catalyzes steps 2–6 within the shikimate pathway [20,21].
In most bacteria, SDH functions as a single monofunctional enzyme. There are two SDH orthologues, AroE
and YdiB, in E. coli, Salmonella typhimurium, Streptococcus pneumoniae, and Haemophilus influenzae. AroE
is strictly specific for shikimate, while YdiB utilizes

either shikimate or quinate as substrate in the shikimate or quinate pathway. However, the complete genome sequence of H. pylori has revealed the only
presence of AroE that plays an essential role in the
metabolism of H. pylori. Recently, the three-dimensional structures of AroE from several bacteria such as
E. coli, Methanococcus jannaschii, and H. influenzae,
and YdiB from E. coli, including structures of
enzyme–cofactor complexes, have been published
[22–25]. All the structures reveal a common fold comprising two domains that are responsible for binding
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 encoding 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 highthroughput screening technology, five novel HpSDH
inhibitors were discovered and their antibacterial activities were also assayed. This study is expected to help
better understand the features of SDH and provide
useful information for the development of novel antibiotics 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

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sequencing result from PCR products, we synthesized
two oligonucleotides for cloning the aroE gene. The
amplified fragment was inserted into the expression
vector pET-22b to generate the recombinant plasmid
pET22b-HpSDH. After confirmed by the sequencing
result from pET22b-HpSDH, the nucleotide sequence
of aroE gene of H. pylori strain SS1 was deposited
into GenBank database under accession number
AY738333. The aroE gene from H. pylori strain SS1 is
a 792-bp fragment (including stop codon) encoding a
polypeptide of 263 amino acids.
Sequence alignment of SDHs from various bacteria
was shown in Fig. 1. Many conserved residues of SDHs

can be found in HpSDH. The conserved residues,
Ser14, Ser16, Lys65, Asn86, Thr101, Asp102, and
Gln244, in the substrate binding site of E. coli SDH
(EcSDH) correspond to the Ser16, Ser18, Lys69,
Asn90, Thr104, Asp105, and Gln237 in HpSDH.
Asn149 and Arg150 of EcSDH are both involved in the

recognition of the adenosine moiety, which are equivalent to Asn148 and Arg149 in HpSDH. Conversely,
HpSDH bears some unique features. There is a glycinerich P-loop with a conserved sequence motif GAGGA
in SDH. As shown in the structure of H. influenzae
SDH, the glycine-rich P-loop determines the interaction
between the enzyme and NADP cofactor [25]. The

Fig. 1. Multiple alignment of SDH sequences from various bacteria. E. coli (SWISS-PROT P15770), H. influenzae (SWISS-PROT P43876),
N. meningitidis (GenBank AAC44905), M. jannaschii (GenBank Q58484), A. fulgidus (GenBank NP_071152), M. tuberculosis (GenBank
NP_217068), and H. pylori (GenBank AAW22052). The conserved sequence motif is underlined, and the strictly conserved residues are
marked with an asterisk. The conserved substitutions are represented by the ‘:’ symbol, and the ‘.’ symbol means that semiconserved substitutions are observed. Alignment was performed by using CLUSTALW program ( />
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H. pylori shikimate dehydrogenase

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

Fig. 2. SDS ⁄ PAGE of the recombinant HpSDH after the purification
procedure. Lane 1, molecular mass marker; lane 2, HpSDH.

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,

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 random 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
from the other SDH crystallographic structures [26],
while the percentage for a-helix of HpSDH is lower

Fig. 3. Molecular mass of the recombinant HpSDH.

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Table 1. Comparison of kinetic parameters of SDH enzymes from
various bacteria. aKinetic parameters for M. tuberculosis SDH are
from [26]. bKinetic parameters for E. coli SDH are from [23]. cKinetic parameters for A. fulgidus SDH are from [27].

SDH
species

Km
Km
kcat ⁄ Km
kcat ⁄ Km
kcat (s)1)
(mM)
(mM)
(M)1s)1)
(M)1s)1)
(shikimate) (shikimate) (NADP) (shikimate) (NADP)

HpSDH
7.7
MtSDHa 399
EcSDHb 237
AfSDHc 361

0.148
0.03
0.065
0.17

0.182
0.063
0.056
0.19

5.2
1.33
3.65
2.12

·
·
·
·

104
107
106
106

3.9
6.33
4.23
1.9

·
·
·
·

104
106
106
106

than that (33%) from the known crystal structures
[26].
Moreover, we have investigated the catalytic properties of HpSDH and the effects of pH and temperature
on HpSDH. The results show that HpSDH has a kcat
of 7.7 ± 0.9 s)1, Km of 0.148 ± 0.028 mm and
kcat ⁄ Km of 5.2 · 104 m)1Ỉs)1 toward shikimate, and a
kcat of 7.1 ± 0.7 s)1, Km of 0.182 ± 0.027 mm and
kcat ⁄ Km of 3.9 · 104 m)1Ỉs)1 toward NADP. Different
from AroE of E. coli [23], HpSDH can oxidize shikimate using NAD as cofactor, which has a kcat of
5.2 ± 0.1 s)1 and Km of 2.9 ± 0.4 mm toward NAD.
HpSDH shows a 10 times higher Km for NAD than
for NADP at saturation of shikimate, suggesting that
NADP is the preferred cofactor of HpSDH. We also
tested whether HpSDH could utilize quinate as substrate. Even in the presence of quinate at a high concentration of 4 mm, HpSDH displayed no activity,
either in the presence of NADP or NAD. In comparison with the kinetic parameters of SDH enzymes from
the other bacteria shown in Table 1 [23,26,27], the Km
values of HpSDH are similar to those of A. fulgidus
SDH (AfSDH), but the kcat value of HpSDH is the
lowest, thus the catalytic efficiency of HpSDH is lower
than those of other SDHs. Notably, the kcat value of

M. tuberculosis SDH (MtSDH) determined by Fonseca
et al. [28] is similar to our result. The low catalytic efficiency of HpSDH may result from the sequence variation in the binding sites of substrate and cofactor.
However, in light of its relative enzyme activity,
HpSDH is still considered as a valuable drug target.
Furthermore, we explored the optimum pH and temperature for HpSDH. As shown in Fig. 4, the enzymatic activity of HpSDH gradually increases between
20 and 60 °C, and decreases from 60 to 80 °C, which
is a similar feature of MtSDH [26]. However, AfSDH
shows its highest activity at or above 95 °C, which
might be due to the organism’s optimal growth temperature at 83 °C [27]. Figure 5 exhibits the pH profile
of HpSDH. It is found that the pH optimum of
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Fig. 4. Temperature profile of HpSDH enzyme activity.

Fig. 5. pH profile of HpSDH enzyme activity.

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 acidic ⁄ 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 acidic ⁄ basic amino acid residues that play crucial roles in
the catalytic process.
HpSDH inhibitor discovery
Using high throughput screening against our constructed chemical library containing 5000 compounds, five
compounds, curcumin (1), 3-(2-naphthyloxy)-4-oxo2-(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 maesaquinone diacetate (5) were

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H. pylori shikimate dehydrogenase

Fig. 6. Chemical structures of compounds 1–5.

discovered as HpSDH inhibitors. Figure 6 shows the
chemical structures of compounds 1–5, and Fig. 7
depicts the dose-dependent inhibition of HpSDH by
these inhibitors. In addition, the inhibitor mode was
also determined. The data collected at varied shikimate
(or NADP) and inhibitor concentrations yielded a series of intersecting lines when plotted as a double-reciprocal plot (Figs 8 and 9). Kinetic analysis indicates
that compounds 1, 2, 3, and 5 are noncompetitive
inhibitors with respect to shikimate as fitted to the
noncompetitive inhibition equation (Eqn 1), where Ki
is the dissociation constant for the inhibitor–enzyme
complex, and aKi is the dissociation constant for the
inhibitor-enzyme–substrate complex. Compound 4 acts
as a competitive inhibitor with respect to shikimate,
fitting to the competitive inhibition equation (Eqn 2).
On the other hand, compounds 1, 4, and 5 are noncompetitive inhibitors, and compounds 2 and 3 are
competitive inhibitors with respect to NADP. Table 2
summarizes the IC50 values and kinetic inhibition data
of compounds 1–5.

Fig. 7. Dose–response curves of HpSDH enzyme inhibition by compounds 1–5. n, 1; d, 2; m, 3; ., 4; and s, 5.

mẳ

ẵS1 ỵ
mẳ

Vmax ẵS
ẵI
aKi ị ỵ Km 1

ỵ ẵIi ị
K

Vmax ẵS


ẵS ỵ Km 1 ỵ ẵIi
K

1ị

2ị

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
strains ATCC 43504 and SS1 in vitro with MIC values
of 16, 16, and 32 lgỈmL)1, respectively. However, no
significant growth inhibition against H. pylori strains
was observed for the other inhibitors, although compounds 3 and 4 show potent inhibitory activities
against HpSDH.

Compound 1, curcumin, is one type of low molecular weight polyphenol derived from the herbal remedy
and dietary spice turmeric. It was reported that curcumin could inhibit the growth of H. pylori in vitro, but
its target was not clear [29]. Compounds 2 and 3 both
belong to chromene derivatives. A possible reason for
the invalidity of compound 3 in H. pylori growth inhibition might be that it bears too large chemical
scaffold to penetrate cell membrane. The invalidity of
compound 4 might result from its poor solubility in
the culture medium.
As shown in Fig. 6, these five inhibitors give four
different types of chemical scaffolds. To date, the
reported SDH inhibitors are almost the dehydroshikimate analogues [30,31]. Therefore, these five discovered
HpSDH inhibitors could obviously present new chemical information that is different from dehydroshikimate analogue, and provide new clues for the
discovery of novel antibacterial agents.

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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 lM (.)]. (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 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 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 (.)].

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Table 2. Inhibition data of the five determined HpSDH inhibitors.
Inhibition mode
Compound

Shikimate

NADP

IC50 (lM)

Ki (lM)

1
2
3
4
5

Noncompetitive
Noncompetitive

Noncompetitive
Competitive
Noncompetitive

Noncompetitive
Competitive
Competitive
Noncompetitive
Noncompetitive

15.4
3.9
13.4
2.9
3.5

5.9
3.9
18.2
1.8
15.4

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 characterized 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 chemical 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 Stratagene (La Jolla, CA, USA). The chemical library containing
5000 compounds was established in our laboratory. All
chemicals were of reagent grade or ultra-pure quality, and
commercially available.

Cloning of H. pylori aroE gene
Based on the genome sequences of H. pylori strains 26695
and J99 (GenBank accession numbers NC_000915 and
NC_000921), two PCR primers (forward: 5¢-CCAAAACG
ATTGGGCTGAAATTG-3¢ and reverse: 5¢-AAAACGCC
CTTTTCTACTAG-3¢) were designed to amplify the
corresponding region including aroE gene on the chromosome of H. pylori strain SS1. The genomic DNA of
H. pylori strain SS1 as a template was prepared by using Genomic DNA Extraction Kit (Sangon, Shanghai, China). The
reaction was performed for 30 cycles: 30 s at 94 °C, 30 s at
55 °C, and 105 s at 72 °C. The amplified DNA segment was
purified and subjected to nucleotide sequencing. According
to the sequencing result, a pair of PCR primers (sense:
5¢-GCGCATCCATATGAAATTAAAATCGTTTGG-3¢ and
antisense: 5¢-CCGCTCGAGAAAAACGCTTCGCATGAC3¢) were synthesized to clone aroE gene from H. pylori strain

±
±
±

±
±

2.2
0.5
3.1
0.2
0.7

±
±
±
±
±

aKi,shikimate (lM)
0.7
0.4
4.3
0.3
1.1

aKi,NADP (lM)

44.5
8.1
28.3
–
58.9

13.6 ± 1.2
–
–
8.0 ± 0.3
11.5 ± 0.7

± 4.2
± 0.9
± 2.6
± 3.7

SS1. The following protocol was conducted for amplification: 94 °C for 30 s, 49 °C for 30 s, and 72 °C for 90 s, 30
cycles. The PCR products were digested with restriction
endonucleases NdeI and XhoI (Takara, Dalian, China), and
cloned into a prokaryotic expression vector pET-22b
(Novagen, Madison, WI, USA) to produce the recombinant
plasmid pET22b-HpSDH containing a C-terminal six-histidine tag for purification purpose. The recombinant clone
pET22b-HpSDH was sequenced and found to be identical
to the sequencing result of PCR products.

Expression and purification of HpSDH
The recombinant clone pET22b-HpSDH was transformed
into E. coli strain BL21(DE3) grown in LB media supplemented with 100 lgỈmL)1 ampicillin at 37 °C. When the
A600 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 fractions 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 °C by
monitoring the reduction of NADP (or NAD) at 340 nm
(e340 ẳ 6180 m)1ặcm)1) in the presence of shikimate. All
assays were conducted in a 96-well microplate spectropho-

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tometer (Tecan GENios reader). The assay mixture (total
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 Km and Vmax values for substrates were determined by varying the concentrations of
one substrate while keeping the other substrate at saturation. In the experiment where shikimate was the varied
substrate (0.0625, 0.125, 0.25, 0.5, and 1 mm), the concentration 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
1 mm). 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 Km and Vmax
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 quinate, 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
the assay solutions contained 2 mm shikimate and 2 mm
NADP. For pH profile analysis, the activity of HpSDH
was measured in different pH buffers (50 mm BisTris ⁄ NaOH for pH 5.0–7.0, Tris ⁄ HCl for pH 8.0–9.0 and
Caps ⁄ NaOH for pH 10–11). As far as the effect of temperature on HpSDH is concerned, the enzymatic activity
assays for HpSDH were processed from 20 to 80 °C. All
the assays were conducted for three times.

Mass spectrometry and CD spectroscopy
The LC ⁄ MS system used for analyzing protein samples was
a combination of HP1100 LC system (Agilent) and LCQDECA mass spectrometer (Thermo Finnigan). The protein
sample was injected into the column by an autosampler
and separated at a low rate of 0.2 mLỈmin)1. The peptide

fraction was detected by PDA (TSP UV6000) and directly
introduced on-line into ESI source. The operating condition
was optimized with standard solution, and the working
parameters of ion source are as follows: capillary temperature 200 °C, spray voltage 5 kV, capillary voltage15 V, and
sheath gas flow rate 20 arbitrary units. The scan mass range
was m ⁄ z 200–2000.
For CD spectral investigation, the solution in 10 mm
phosphate buffer (pH 7.5) of 10 lm HpSDH was prepared
by dialysis. All the CD spectral measurements were carried
out by a JASCO J-810 spectropolarimeter with a 1-mm
path-length cuvette at 25 °C. Experimental data were corrected by subtracting the blank obtained under the same
conditions in the absence of protein. The CD measurement
of HpSDH was repeated three times.

4690

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 initial 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. IC50 values of compounds 1–5 were obtained by fitting the data to a sigmoid dose–response equation of the
origin software (OriginLab, Northampton, MA, USA).
Afterwards, inhibitor modality was determined by measuring 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,
0.125, 0.25, 0.5, and 1 mm) when the concentration of
inhibitor was varied from 0 to 20 lm.

Antibiotic susceptibility test
The MIC (minimal inhibitory concentration) of HpSDH
inhibitor identified by the above-mentioned high-throughput screening was determined by the standard agar dilution
method using Columbia agar supplemented with 10%
sheep blood containing two-fold serial dilutions of agents.
H. pylori strains ATCC 43504 and SS1 were used as tested
bacteria. The plates were inoculated with a bacterial suspension (108 cfu ⁄ mL) in sterile saline with a multipoint inoculator (Sakuma Seisakusho, Tokyo, Japan). Compoundfree Columbia agar media were used as black controls, and
Columbia agar media containing tetracycline were applied
as positive controls. Inoculated plates were incubated at
37 °C under microaerobic conditions and examined after
3 days. The MIC was defined as the lowest concentration
of antimicrobial agent that completely inhibited visible
bacterial growth.

Acknowledgements
This work was supported by the State Key Program
of Basic Research of China (grants 2002CB512807,

FEBS Journal 273 (2006) 4682–4692 ª 2006 The Authors Journal compilation ª 2006 FEBS


C. Han et al.

2004CB58905), the National Natural Science Foundation of China (grants 30525024 and 20372069), Shanghai Basic Research Project from the Shanghai Science
and Technology Commission (grant 054319908).

References
1 Warren JR & Marshall BJ (1983) Unidentified curved
bacilli on gastric epithelium in active chronic gastritis.
Lancet 1, 1273–1275.
2 Brown LM (2000) Helicobacter pylori: epidemiology and
routes of transmission. Epidemiol Rev 22, 283–297.
3 Cover TL & Blaser MJ (1996) Helicobacter pylori infection, a paradigm for chronic mucosal inflammation:
pathogenesis and implications for eradication and prevention. Adv Intern Med 41, 85–117.
4 Ulmer HJ, Beckerling A & Gatz G (2003) Recent use of
proton pump inhibitor-based triple therapies for the eradication of H. pylori: a broad data review. Helicobacter
8, 95–104.
5 Cameron EA, Powell KU, Baldwin L, Jones P, Bell GD
& Williams SG (2004) Helicobacter pylori: antibiotic
resistance and eradication rates in Suffolk, UK, 1991–
2001. J Med Microbiol 53, 535–538.
6 Tomb JF, White O, Kerlavage AR, Clayton RA, Sutton
GG, Fleischmann RD, Ketchum KA, Klenk HP, Gill S,
Dougherty BA et al. (1997) The complete genome
sequence of the gastric pathogen Helicobacter pylori.
Nature 388, 539–547.
7 Alm RA, Ling LS, Moir DT, King BL, Brown ED,

Doig PC, Smith DR, Noonan B, Guild BC, deJonge BL
et al. (1999) Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter
pylori. Nature 397, 176–180.
8 Dean PM, Zanders ED & Bailey DS (2001) Industrialscale, genomics-based drug design and discovery. Trends
Biotechnol 19, 288–292.
9 McDevitt D & Rosenberg M (2001) Exploiting genomics to discover new antibiotics. Trends Microbiol 9,
611–617.
10 Parish T & Stoker NG (2002) The common aromatic
amino acid biosynthesis pathway is essential in Mycobacterium tuberculosis. Microbiology 148, 3069–3077.
11 Kishore GM & Shah DM (1988) Amino acid biosynthesis inhibitors as herbicides. Annu Rev Biochem 57, 627–
663.
12 Roberts F, Roberts CW, Johnson JJ, Kyle DE, Krell T,
Coggins JR, Coombs GH, Milhous WK, Tzipori S,
Ferguson DJ et al. (1998) Evidence for the shikimate
pathway in apicomplexan parasites. Nature 393, 801–805.
13 Coggins JR, Abell C, Evans LB, Frederickson M,
Robinson DA, Roszak AW & Lapthorn AP (2003)
Experiences with the shikimate-pathway enzymes as targets for rational drug design. Biochem Soc Trans 31,
548–552.

H. pylori shikimate dehydrogenase

14 Bornemann S, Ramjee MK, Balasubramanian S, Abell
C, Coggins JR, Lowe DJ & Thorneley RN (1995)
Escherichia coli chorismate synthase catalyzes the
conversion of (6S) -6-fluoro-5-enolpyruvylshikimate3-phosphate to 6-fluorochorismate. J Biol Chem 270,
22811–22815.
15 Davies GM, Barrett-Bee KJ, Jude DA, Lehan M,
Nichols WW, Pinder PE, Thain JL, Watkins WJ &
Wilson RG (1994) (6S)-6-Fluoroshikimic acid, an

antibacterial agent acting on the aromatic biosynthetic
pathway. Antimicrob Agents Chemother 38, 403–406.
16 Gonzalez-Bello C, Lence E, Toscano MD, Castedo L,
Coggins JR & Abell C (2003) Parallel solid-phase
synthesis and evaluation of inhibitors of Streptomyces
coelicolor type II dehydroquinase. J Med Chem 46,
5735–5744.
17 Thomas MG, Lawson C, Allanson NM, Leslie BW,
Bottomley JR, McBride A & Olusanya OA (2003) A
series of 2(Z)-2-benzylidene-6,7-dihydroxybenzofuran3[2H]-ones as inhibitors of chorismate synthase. Bioorg
Med Chem Lett 13, 423–426.
18 Bonner CA & Jensen RA (1994) Cloning of cDNA
encoding the bifunctional dehydroquinasshikimate
dehydrogenase of aromatic-amino-acid biosynthesis in
Nicotiana tabacum. Biochem J 302, 11–14.
19 Deka RK, Anton IA, Dunbar B & Coggins JR (1994)
The characterisation of the shikimate pathway enzyme
dehydroquinase from Pisum sativum. FEBS Lett 349,
397–402.
20 Charles IG, Keyte JW, Brammar WJ, Smith M &
Hawkins AR (1986) The isolation and nucleotide
sequence of the complex AROM locus of Aspergillus
nidulans. Nucleic Acids Res 14, 2201–2213.
21 Duncan K, Edwards RM & Coggins JR (1987) The
pentafunctional arom enzyme of Saccharomyces cerevisiae is a mosaic of monofunctional domains. Biochem J
246, 375–386.
22 Benach J, Lee I, Edstrom W, Kuzin AP, Chiang Y,
˚
Acton TB, Montelione GT & Hunt JF (2003) The 2.3-A
crystal structure of the shikimate 5-dehydrogenase

orthologue YdiB from Escherichia coli suggests a novel
catalytic environment for an NAD-dependent dehydrogenase. J Biol Chem 278, 19176–19182.
23 Michel G, Roszak AW, Sauve V, Maclean J, Matte A,
Coggins JR, Cygler M & Lapthorn AJ (2003) Structures
of shikimate dehydrogenase AroE and its paralog YdiB.
J Biol Chem 278, 19463–19472.
24 Padyana AK & Burley SK (2003) Crystal structure of
shikimate 5-dehydrogenase (SDH) bound to NADP:
insights into function and evolution. Structure 11, 1005–
1013.
25 Ye S, von Delft F, Brooun A, Knuth MW, Swanson
RV & McRee DE (2003) The crystal structure of shikimate dehydrogenase (AroE) reveals a unique NADPH
binding mode. J Bacteriol 185, 4144–4151.

FEBS Journal 273 (2006) 4682–4692 ª 2006 The Authors Journal compilation ª 2006 FEBS

4691


H. pylori shikimate dehydrogenase

C. Han et al.

26 Zhang X, Zhang S, Hao F, Lai XYuH, Huang Y &
Wang H (2005) Expression, purification and properties
of shikimate dehydrogenase from Mycobacterium tuberculosis. J Biochem Mol Biol 38, 624–631.
27 Lim S, Schroder I & Monbouquette HG (2004) A thermostable shikimate 5-dehydrogenase from the archaeon
Archaeoglobus fulgidus. FEMS Microbiol Lett 238, 101–
106.
28 Fonseca IO, Magalhaes ML, Oliveira JS, Silva RG,

Mendes MA, Palma MS, Santos DS & Basso LA (2006)
Functional shikimate dehydrogenase from Mycobacterium tuberculosis H37Rv: purification and characterization. Protein Expr Purif 46, 429–437.

4692

29 Mahady GB, Pendland SL, Yun G & Lu ZZ (2002)
Turmeric (Curcuma longa) and curcumin inhibit the
growth of Helicobacter pylori, a group 1 carcinogen.
Anticancer Res 22, 4179–4181.
30 Baillie AC, Corbett JR, Dowsett JR & McCloskey P
(1972) Inhibitors of shikimate dehydrogenase as potential herbicides. Biochem J 126, 21P.
31 Dowsett JR, Middleton B, Corbett JR & Tubbs PK
(1972) The anomalous inhibition of shikimate dehydrogenase by analogues of dehydroshikimate. Biochim
Biophys Acta 276, 344–349.

FEBS Journal 273 (2006) 4682–4692 ª 2006 The Authors Journal compilation ª 2006 FEBS