pylori is a major causative factor for several gastrointestinal illnesses, including gastritis, Keywords antibacterial agent; drug target; enzyme inhibition; Helicobacter pylori; shikima
Trang 1of 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
Helicobacter pyloriis 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
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: xshen@mail.shcnc.ac.cn,
hjiang@mail.shcnc.ac.cn,
simmkulh@mail.shcnc.ac.cn
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 shiki-mate pathway It catalyzes the NADPH-dependent reduction of 3-dehy-droshikimate 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 recom-binant 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 60C 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 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 inhibi-tion pattern, and compound 4 displays competitive inhibiinhibi-tion pattern with respect to shikimate Compounds 1, 4, and 5 display noncompetitive inhibi-tion mode, and compounds 2 and 3 show competitive inhibiinhibi-tion 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 lgÆmL)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
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.
Trang 2peptic 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
metro-nidazole, 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
resul-ted 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
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
have been prepared to investigate the mechanism of
the enzymes within the shikimate pathway [16,17]
Shikimate dehydrogenase (SDH, EC 1.1.1.25)
cata-lyzes the fourth reaction in the shikimate pathway,
and is responsible for the NADPH-dependent
reduc-tion of 3-dehydroshikimate to shikimate SDH belongs
to the superfamily of NAD(P)H-dependent oxidore-ductase In plants, including Pisum sativum and Nicoti-ana tabacum, SDH is associated with 3-dehydroquinate dehydratase to form bifunctional enzyme [18,19] In fungi and yeast, such as Aspergillus nidulans and Sac-charomyces cerevisiae, SDH exists as a component of the penta-functional AROM enzyme complex that cat-alyzes steps 2–6 within the shikimate pathway [20,21]
In most bacteria, SDH functions as a single monofunc-tional enzyme There are two SDH orthologues, AroE and YdiB, in E coli, Salmonella typhimurium, Strepto-coccus pneumoniae, and Haemophilus influenzae AroE
is strictly specific for shikimate, while YdiB utilizes either shikimate or quinate as substrate in the shiki-mate or quinate pathway However, the complete gen-ome 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-dimen-sional 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 com-prising 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 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
Trang 3sequencing 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 equival-ent to Asn148 and Arg149 in HpSDH Conversely, HpSDH bears some unique features There is a glycine-rich 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 sub-stitutions are observed Alignment was performed by using CLUSTALW program (http://www.ebi.ac.uk/clustalw/index.html).
Trang 4alanine 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 tuberculosisSDH, 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 from the other SDH crystallographic structures [26], while the percentage for a-helix of HpSDH is lower
Fig 2 SDS ⁄ PAGE of the recombinant HpSDH after the purification
procedure Lane 1, molecular mass marker; lane 2, HpSDH.
Fig 3 Molecular mass of the recombinant HpSDH.
Trang 5than that (33%) from the known crystal structures
[26]
Moreover, we have investigated the catalytic
proper-ties 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)1Æs)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)1Æs)1 toward NADP Different
from AroE of E coli [23], HpSDH can oxidize
shiki-mate 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
sub-strate Even in the presence of quinate at a high
con-centration of 4 mm, HpSDH displayed no activity,
either in the presence of NADP or NAD In
compar-ison 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 tuberculosisSDH (MtSDH) determined by Fonseca
et al.[28] is similar to our result The low catalytic
effi-ciency of HpSDH may result from the sequence
vari-ation 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
tem-perature for HpSDH As shown in Fig 4, the
enzy-matic activity of HpSDH gradually increases between
20 and 60C, and decreases from 60 to 80 C, which
is a similar feature of MtSDH [26] However, AfSDH
shows its highest activity at or above 95C, which
might be due to the organism’s optimal growth
tem-perature at 83C [27] Figure 5 exhibits the pH profile
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. aKinetic 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
species
k cat (s)1)
(shikimate)
Km (m M ) (shikimate)
Km (m M ) (NADP)
kcat⁄ K m
( M )1s)1)
(shikimate)
kcat⁄ K m
( M )1s)1)
(NADP)
6.33 · 10 6
EcSDH b 237 0.065 0.056 3.65 · 10 6 4.23 · 10 6
Fig 4 Temperature profile of HpSDH enzyme activity.
Fig 5 pH profile of HpSDH enzyme activity.
Trang 6discovered 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
ser-ies of intersecting lines when plotted as a
double-recip-rocal 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
non-competitive inhibitors, and compounds 2 and 3 are
competitive inhibitors with respect to NADP Table 2
summarizes the IC50values and kinetic inhibition data
of compounds 1–5
m¼ Vmax½S
½Sð1 þaK½I
iÞ þ Kmð1 þ½IK
iÞ ð1Þ
m¼ Vmax½S
½S þ Km 1þ½IK
i
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 lgÆmL)1, respectively However, no significant growth inhibition against H pylori strains was observed for the other inhibitors, although com-pounds 3 and 4 show potent inhibitory activities against HpSDH
Compound 1, curcumin, is one type of low molecu-lar weight polyphenol derived from the herbal remedy and dietary spice turmeric It was reported that curcu-min 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 inhi-bition 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 dehydroshiki-mate analogues [30,31] Therefore, these five discovered HpSDH inhibitors could obviously present new chem-ical information that is different from dehydroshiki-mate analogue, and provide new clues for the discovery of novel antibacterial agents
Fig 6 Chemical structures of compounds 1–5.
Fig 7 Dose–response curves of HpSDH enzyme inhibition by
com-pounds 1–5 n, 1; d, 2; m, 3; , 4; and s, 5.
Trang 7Fig 8 Inhibition of HpSDH toward shikimate by increasing concentrations of compounds 1–5 (A) Compound 1 [0 l M (n), 5 l M (d), 10 l M
(m), and 20 l M (.)] (B) Compound 2 [0 l M (n), 2.5 l M (d), 5 l M (m), and 10 l M (.)] (C) Compound 3 [0 l M (n), 5 l M (d), 10 l M (m), and
20 l M (.)] (D) Compound 4 [0 l M (n), 1 l M (d), 2.5 l M (m), and 5 l M (.)] (E) Compound 5 [0 l M (n), 1 l M (d), 5 l M (m), and 10 l M (.)].
Fig 9 Inhibition of HpSDH toward NADP by increasing concentrations of compounds 1–5 (A) Compound 1 [0 l M (n), 2.5 l M (d), 5 l M (m), and 10 l M (.)] (B) Compound 2 [0 l M (n), 2.5 l M (d), 5 l M (m), and 10 l M (.)] (C) Compound 3 [0 l M (n), 5 l M (d), 10 l M (m), and 20 l M
(.)] (D) Compound 4 [0 l M (n), 1 l M (d), 2.5 l M (m), and 5 l M (.)] (E) Compound 5 [0 l M (n), 2.5 l M (d), 5 l M (m), and 10 l M (.)].
Trang 8In 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 pyloristrains SS1 and ATCC 43504 were obtained from
Shanghai Institute of Digestive Disease (Shanghai, China)
E colihost strain BL21(DE3) was purchased from
Strata-gene (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
corresponding region including aroE gene on the
chromo-some of H pylori strain SS1 The genomic DNA of
H pyloristrain SS1 as a template was prepared by using
Ge-nomic DNA Extraction Kit (Sangon, Shanghai, China) The
reaction was performed for 30 cycles: 30 s at 94C, 30 s at
55C, 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¢-CCGCTCGAGAAAAACGCTTCGCATGAC-3¢) were synthesized to clone aroE gene from H pylori strain
SS1 The following protocol was conducted for amplifica-tion: 94C 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-histi-dine 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 supple-mented with 100 lgÆmL)1 ampicillin at 37C When the
A600 reached 0.6, the culture was induced by 0.4 mm IPTG and incubated at 25C for additional 6 h The cells were harvested by centrifugation and suspended in
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 4C Protein concentration was determined by Bradford assay using bovine serum albumin as standard
Enzymatic activity assay
The enzymatic activity of HpSDH was assayed at 25C by monitoring the reduction of NADP (or NAD) at 340 nm (e340¼ 6180 m)1Æcm)1) in the presence of shikimate All assays were conducted in a 96-well microplate
spectropho-Table 2 Inhibition data of the five determined HpSDH inhibitors.
Compound
Inhibition mode
IC50(l M ) Ki(l M ) aKi,shikimate(l M ) aKi,NADP(l M )
Trang 9tometer (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
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
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 Kmand 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
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
the assay solutions contained 2 mm shikimate and 2 mm
NADP For pH profile analysis, the activity of HpSDH
Bis-Tris⁄ 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
tem-perature on HpSDH is concerned, the enzymatic activity
assays for HpSDH were processed from 20 to 80C 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
LCQ-DECA mass spectrometer (Thermo Finnigan) The protein
sample was injected into the column by an autosampler
and separated at a low rate of 0.2 mLÆmin)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
tempera-ture 200C, 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 25C Experimental data were
cor-rected by subtracting the blank obtained under the same
conditions in the absence of protein The CD measurement
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 IC50values of compounds 1–5 were obtained by fit-ting the data to a sigmoid dose–response equation of the
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, 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-through-put 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 sus-pension (108cfu⁄ mL) in sterile saline with a multipoint ino-culator (Sakuma Seisakusho, Tokyo, Japan) Compound-free Columbia agar media were used as black controls, and Columbia agar media containing tetracycline were applied
as positive controls Inoculated plates were incubated at
37C 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,
Trang 102004CB58905), the National Natural Science
Founda-tion of China (grants 30525024 and 20372069),
Shang-hai Basic Research Project from the ShangShang-hai Science
and Technology Commission (grant 054319908)
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