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 Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China 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: 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 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 demonstrate noncompetitive inhibition pattern, and compound displays competitive inhibition pattern with respect to shikimate Compounds 1, 4, and display noncompetitive inhibition mode, and compounds and show competitive inhibition mode with respect to NADP Antibacterial assays demonstrate that compounds 1, 2, and 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 4682 FEBS Journal 273 (2006) 4682–4692 ª 2006 The Authors Journal compilation ª 2006 FEBS 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 FEBS Journal 273 (2006) 4682–4692 ª 2006 The Authors Journal compilation ª 2006 FEBS 4683 H pylori shikimate dehydrogenase C Han et al 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 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 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 (http://www.ebi.ac.uk/clustalw/index.html) 4684 FEBS Journal 273 (2006) 4682–4692 ª 2006 The Authors Journal compilation ª 2006 FEBS C Han et al 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 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 Molecular mass of the recombinant HpSDH FEBS Journal 273 (2006) 4682–4692 ª 2006 The Authors Journal compilation ª 2006 FEBS 4685 H pylori shikimate dehydrogenase C Han et al Table 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 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 [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 exhibits the pH profile of HpSDH It is found that the pH optimum of 4686 Fig Temperature profile of HpSDH enzyme activity Fig pH profile of HpSDH enzyme activity HpSDH is between 8.0 and 9.0, and the pH optimum of AfSDH is between 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 FEBS Journal 273 (2006) 4682–4692 ª 2006 The Authors Journal compilation ª 2006 FEBS C Han et al H pylori shikimate dehydrogenase Fig Chemical structures of compounds 1–5 discovered as HpSDH inhibitors Figure shows the chemical structures of compounds 1–5, and Fig 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 and 9) Kinetic analysis indicates that compounds 1, 2, 3, and 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 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 are noncompetitive inhibitors, and compounds and are competitive inhibitors with respect to NADP Table summarizes the IC50 values and kinetic inhibition data of compounds 1–5 Fig Dose–response curves of HpSDH enzyme inhibition by compounds 1–5 n, 1; d, 2; m, 3; , 4; and s, mẳ ẵS1 ỵ mẳ Vmax ẵS ẵI aKi ị ỵ Km ỵ ẵIi ị K Vmax ẵS   ẵS ỵ Km 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 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 and 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 and both belong to chromene derivatives A possible reason for the invalidity of compound in H pylori growth inhibition might be that it bears too large chemical scaffold to penetrate cell membrane The invalidity of compound 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 FEBS Journal 273 (2006) 4682–4692 ª 2006 The Authors Journal compilation ª 2006 FEBS 4687 H pylori shikimate dehydrogenase C Han et al Fig Inhibition of HpSDH toward shikimate by increasing concentrations of compounds 1–5 (A) Compound [0 lM (n), lM (d), 10 lM (m), and 20 lM (.)] (B) Compound [0 lM (n), 2.5 lM (d), lM (m), and 10 lM (.)] (C) Compound [0 lM (n), lM (d), 10 lM (m), and 20 lM (.)] (D) Compound [0 lM (n), lM (d), 2.5 lM (m), and lM (.)] (E) Compound [0 lM (n), lM (d), lM (m), and 10 lM (.)] Fig Inhibition of HpSDH toward NADP by increasing concentrations of compounds 1–5 (A) Compound [0 lM (n), 2.5 lM (d), lM (m), and 10 lM (.)] (B) Compound [0 lM (n), 2.5 lM (d), lM (m), and 10 lM (.)] (C) Compound [0 lM (n), lM (d), 10 lM (m), and 20 lM (.)] (D) Compound [0 lM (n), lM (d), 2.5 lM (m), and lM (.)] (E) Compound [0 lM (n), 2.5 lM (d), lM (m), and 10 lM (.)] 4688 FEBS Journal 273 (2006) 4682–4692 ª 2006 The Authors Journal compilation ª 2006 FEBS C Han et al H pylori shikimate dehydrogenase Table Inhibition data of the five determined HpSDH inhibitors Inhibition mode Compound Shikimate NADP IC50 (lM) Ki (lM) 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 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, 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 °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 ¼ 6180 m)1Ỉcm)1) in the presence of shikimate All assays were conducted in a 96-well microplate spectropho- FEBS Journal 273 (2006) 4682–4692 ª 2006 The Authors Journal compilation ª 2006 FEBS 4689 H pylori shikimate dehydrogenase C Han et al 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 mm), the concentration of NADP was maintained at mm, whereas the concentration of shikimate was fixed at mm when NADP was the varied substrate (0.0625, 0.125, 0.25, 0.5, and 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 mm when NAD was the varied substrate (0.25, 0.5, 1, 2, and 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), mm quinate, and 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 mm shikimate and 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 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), mm shikimate, and 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 mm, shikimate was a varied substrate (0.0625, 0.125, 0.25, 0.5, and mm) when the concentration of inhibitor was varied from to 20 lm In parallel, in the inhibition experiment where the shikimate concentration was fixed at mm, NADP was a varied substrate (0.0625, 0.125, 0.25, 0.5, and mm) when the concentration of inhibitor was varied from 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 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 Warren JR & Marshall BJ (1983) Unidentified curved bacilli on gastric epithelium in active chronic gastritis Lancet 1, 1273–1275 Brown LM (2000) Helicobacter pylori: epidemiology and routes of transmission Epidemiol Rev 22, 283–297 Cover TL & Blaser MJ (1996) Helicobacter pylori infection, a paradigm for chronic mucosal 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Methanococcus jannaschii, and H influenzae, and YdiB from E coli, including structures of. .. 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