Abstract
Aim:
To investigate the inhibition features of the natural product juglone (5-hydroxy-1,4-naphthoquinone) against the three key enzymes from Helicobacter pylori (cystathionine γ-synthase [HpCGS], malonyl-CoA:acyl carrier protein transacylase [HpFabD], and β-hydroxyacyl-ACP dehydratase [HpFabZ]).
Methods:
An enzyme inhibition assay against HpCGS was carried out by using a continuous coupled spectrophotometric assay approach. The inhibition assay of HpFabD was performed based on the α-ketoglutarate dehydrogenase-coupled system, while the inhibition assay for HpFabZ was monitored by detecting the decrease in absorbance at 260 nm with crotonoyl-CoA conversion to β-hydroxybutyryl-CoA. The juglone/FabZ complex crystal was obtained by soaking juglone into the HpFabZ crystal, and the X-ray crystal structure of the complex was analyzed by molecular replacement approach.
Results:
Juglone was shown to potently inhibit HpCGS, HpFabD, and HpFabZ with the half maximal inhibitory concentration IC50 values of 7.0±0.7, 20±1, and 30±4 μmol/L, respectively. An inhibition-type study indicated that juglone was a non-competitive inhibitor of HpCGS against O-succinyl-L-homoserine (Ki=αKi=24 μmol/L), an uncompetitive inhibitor of HpFabD against malonyl-CoA (αKi=7.4 μmol/L), and a competitive inhibitor of HpFabZ against crotonoyl-CoA (Ki=6.8 μmol/L). Moreover, the crystal structure of the HpFabZ/juglone complex further revealed the essential binding pattern of juglone against HpFabZ at the atomic level.
Conclusion:
HpCGS, HpFabD, and HpFabZ are potential targets of juglone.
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Abbreviations
- IC50:
-
the half maximal inhibitory concentration
- K i :
-
the dissociation constant for inhibitor binding
- CoA:
-
coenzyme A
- ACP:
-
acyl carrier protein transacylase
- PDB:
-
Protein Data Bank
- HO-HxoDH:
-
D-2-Hydroxyisocaproate dehydrogenase
References
Dubreuil JD, Giudice GD, Rappuoli R . Helicobacter pylori interactions with host serum and extracellular matrix proteins: potential role in the infectious process. Microbiol Mol Biol Rev 2002; 66: 617–29.
Cover TL, Blaser MJ . Helicobacter pylori infection, a paradigm for chronic mucosal inflammation: pathogenesis and implications for eradication and prevention. Adv Intern Med 1996; 41: 85–117.
Cameron EA, Powell KU, Baldwin L, Jones P, Bell GD, Williams SG . Helicobacter pylori: antibiotic resistance and eradication rates in Suffolk, UK, 1991 2001. J Med Microbiol 2004; 53: 535–8.
Paulsen MT, Ljungman M . The natural toxin juglone causes degradation of p53 and induces rapid H2AX phosphorylation and cell death in human fibroblasts. Toxicol Appl Pharmacol 2005; 209: 1–9.
Inbaraj JJ, Chignell CF . Cytotoxic action of juglone and plumbagin: a mechanistic study using HaCaT keratinocytes. Chem Res Toxicol 2004; 17: 55–62.
Rippmann JF, Hobbie S, Daiber C, Guilliard B, Bauer M, Birk J, et al. Phosphorylation-dependent proline isomerization catalyzed by Pin1 is essential for tumor cell survival and entry into mitosis. Cell Growth Differ 2000; 11: 409–16.
Chao SH, Greenleaf AL, Price DH . Juglone, an inhibitor of the peptidyl-prolyl isomerase Pin1, also directly blocks transcription. Nucleic Acids Res 2001; 29: 767–73.
Varga Z, Bene L, Pieri C, Damjanovich S, Gaspar R . The effect of juglone on the membrane potential and whole-cell K+ currents of human lymphocytes. Biochem Biophys Res Commun 1996; 218: 828–32.
Hennig L, Christner C, Kipping M, Schelbert B, Rucknagel KP, Grabley S, et al. Selective inactivation of parvulin-like peptidyl-prolylcis/trans isomerases by juglone. Biochemistry 1998; 37: 5953–60.
Alice MC, Tannis MJ, Charles DH . Antimicrobial activity of juglone. Phytotherapy Research 1990; 4: 11–4.
Clausen T, Huber R, Prade L, Wahl MC, Messerschmidt A . Crystal structure of Escherichia coli cystathionine gamma-synthase at 1.5 A resolution. EMBO J 1998; 17: 6827–38.
Soda K . Microbial sulfur amino acids: an overview. Methods Enzymol 1987; 143: 453 9.
Aitken SM, Kim DH, Kirsch JF . Escherichia coli cystathionine gamma-synthase does not obey ping-pong kinetics. Novel continuous assays for the elimination and substitution reactions. Biochemistry 2003; 42: 11297–306.
Wahl MC, Huber R, Prade L, Marinkovic S, Messerschmidt A, Clausen T . Cloning, purification, crystallization, and preliminary X-ray diffraction analysis of cystathionine gamma-synthase from E coli. FEBS Lett 1997; 414: 492–6.
Salama NR, Shepherd B, Falkow S . Global transposon mutagenesis and essential gene analysis of Helicobacter pylori. J Bacteriol 2004; 186: 7926–35.
Campbell JW, Cronan JE . Bacterial fatty acid biosynthesis: targets for antibacterial drug discovery. Annu Rev Microbiol 2001; 55: 305–32.
White SW, Zheng J, Zhang YM, Rock CO . The structure biology of type II fatty acid biosynthesis. Annu Rev Biochemistry 2005; 74: 791–831.
Magnuson K, Jackowski S, Rock CO, Cronan JE . Regulation of fatty acid biosynthesis in Escherichia coli. Microbiol Rev 1993; 57: 522–42.
Williamson IP, Wakil SJ . Studies on the mechanism of fatty acid synthesis. XVII. Preparation and general properties of acetyl coenzyme A and malonyl coenzyme A-acyl carrier protein transacylases. J Biol Chem 1966; 241: 2326–32.
Ruch FE, Vagelos PR . The isolation and general properties of Escherichia coli malonyl coenzyme A-acyl carrier protein transacylase. J Biol Chem 1973; 248: 8086–94.
Verwoert II, Verbree EC, van der Linden KH, Nijkamp HJ, Stuitje AR . Cloning, nucleotide sequence, and expression of the Escherichia coli fabD gene, encoding malonyl coenzyme A-acyl carrier protein transacylase. J Bacteriol 1992; 174: 2851–7.
Kutchma AJ, Hoang TT, Schweizer HP . Characterization of a Pseudomonas aeruginosa fatty acid biosynthetic gene cluster: purification of acyl carrier protein (ACP) and malonyl-coenzyme A:ACP transacylase (FabD). J Bacteriol 1999; 181: 5498–504.
Mohan S, Kelly TM, Eveland SS, Raetz CR, Anderson MS . An Escherichia coli gene (FabZ) encoding (3R)-hydroxymyristoyl acyl carrier protein dehydrase. Relation to fabA and suppression of mutations in lipid A biosynthesis. J Biol Chem 1994; 269: 32896–903.
Heath RJ, Rock CO . Roles of the FabA and FabZ beta-hydroxyacylacyl carrier protein dehydratases in Escherichia coli fatty acid biosynthesis. J Biol Chem 1996; 271: 27795–801.
Pillai S, Rajagopal C, Kapoor M, Kumar G, Gupta A, Surolia N . Functional characterization of beta-ketoacyl-ACP reductase (FabG) from Plasmodium falciparum. Biochem Biophys Res Commun 2003; 303: 387–92.
Sharma SK, Kapoor M, Ramya TN, Kumar S, Kumar G, Modak R, et al. Identification, characterization, and inhibition of Plasmodium falciparum beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ). J Biol Chem 2003; 278: 45661–71.
Kong YH, Wu DL, Bai HY, Han C, Chen J, Chen LL, et al. Enzymatic characterization and inhibitor discovery of a new cys-tathionine γ-synthase (CGS) from Helicobacter pylori. J Biochem (Tokyo) 2008; 143: 59–68.
Liu WZ, Han C, Hu LH, Chen KX, Shen X, Jiang HL . Characterization and inhibitor discovery of one novel malonyl-CoA: acyl carrier protein transacylase (MCAT) from Helicobacter pylori. FEBS Lett 2006; 580: 697–702.
Liu WZ, Luo C, Han C, Peng SY, Yang Y, Yue J, et al. A new beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from Helicobacter pylori: molecular cloning, enzymatic characterization, and structural modeling. Biochem Biophys Res Commun 2005; 333: 1078–86.
Zhang L, Liu WZ, Hu TC, Du L, Luo C, Chen KX, et al. Structural basis for catalytic and inhibitory mechanisms of beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ). J Biol Chem 2008; 283: 5370–9.
Chen LL, Gui CS, Luo XM, Yang QG, Gunther S, Scandella E, et al. Cinanserin is an inhibitor of the 3C-like proteinase of severe acute respiratory syndrome coronavirus and strongly reduces virus replication in vitro. J Virol 2005; 79: 7095–103.
Otwinowski Z, Minor W . Methods in Enzymology 1997; 276: 307–26.
Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve, et al. Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 1998; 54: 905–21.
Emsley P, Cowtan K . Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 2004; 60: 2126–32.
Silver LL . Multi-targeting by monotherapeutic antibacterials. Nat Rev Drug Discov 2007; 6: 41–55.
Inatsu S, Ohsaki A, Nagata K . Idebenone acts against growth of Helicobacter pylori by inhibiting its respiration. Antimicrob Agents Chemother 2006; 50: 2237–9.
Park BS, Lee HK, Lee SE, Piao XL, Takeoka GR, Wong RY, et al. Antibacterial activity of Tabebuia impetiginosa Martius ex DC (Taheebo) against Helicobacter pylori. J Ethnopharmacol 2006; 105: 255–62.
Tasdemir D, Lack G, Brun R, Ruedi P, Scapozza L, Perozzo R . Inhibition of Plasmodium falciparum fatty acid biosynthesis: evaluation of FabG, FabZ, and FabI as drug targets for flavonoids. J Med Chem 2006; 49: 3345–53.
Wang J, Soisson SM, Young K, Shoop W, Kodali S, Galgoci A, et al. Platensimycin is a selective FabF inhibitor with potent antibiotic properties. Nature 2006; 441: 358–61.
Delanoue WL . The PyMOL Molecular Graphics System. San Carlos, CA: DelanoScientific, 2000.
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This work was supported by the National Natural Science Foundation of China (No 30525024, 20721003, and 90713046).
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Kong, Yh., Zhang, L., Yang, Zy. et al. Natural product juglone targets three key enzymes from Helicobacter pylori: inhibition assay with crystal structure characterization. Acta Pharmacol Sin 29, 870–876 (2008). https://doi.org/10.1111/j.1745-7254.2008.00808.x
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DOI: https://doi.org/10.1111/j.1745-7254.2008.00808.x
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