Natural product juglone targets three key enzymes from Helicobacter pylori: inhibition assay with crystal structure characterization

AbstractAim: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.

[1]  A. Stuitje,et al.  Cloning, nucleotide sequence, and expression of the Escherichia coli fabD gene, encoding malonyl coenzyme A-acyl carrier protein transacylase , 1992, Journal of bacteriology.

[2]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[3]  S. Damjanovich,et al.  The effect of juglone on the membrane potential and whole-cell K+ currents of human lymphocytes. , 1996, Biochemical and biophysical research communications.

[4]  J. Kirsch,et al.  Escherichia coli cystathionine gamma-synthase does not obey ping-pong kinetics. Novel continuous assays for the elimination and substitution reactions. , 2003, Biochemistry.

[5]  Hualiang Jiang,et al.  Enzymatic characterization and inhibitor discovery of a new cystathionine {gamma}-synthase from Helicobacter pylori. , 2007, Journal of biochemistry.

[6]  R. Wong,et al.  Antibacterial activity of Tabebuia impetiginosa Martius ex DC (Taheebo) against Helicobacter pylori. , 2006, Journal of ethnopharmacology.

[7]  H. Schweizer,et al.  Characterization of a Pseudomonas aeruginosa Fatty Acid Biosynthetic Gene Cluster: Purification of Acyl Carrier Protein (ACP) and Malonyl-Coenzyme A:ACP Transacylase (FabD) , 1999, Journal of bacteriology.

[8]  Kaixian Chen,et al.  Characterization and inhibitor discovery of one novel malonyl‐CoA: Acyl carrier protein transacylase (MCAT) from Helicobacter pylori , 2006, FEBS letters.

[9]  R. Rappuoli,et al.  Helicobacter pylori Interactions with Host Serum and Extracellular Matrix Proteins: Potential Role in the Infectious Process , 2002, Microbiology and Molecular Biology Reviews.

[10]  I. P. Williamson,et al.  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. , 1966, The Journal of biological chemistry.

[11]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[12]  A. Clark,et al.  Antimicrobial activity of juglone , 1990 .

[13]  S. Sharma,et al.  Identification, Characterization, and Inhibition of Plasmodium falciparum β-Hydroxyacyl-Acyl Carrier Protein Dehydratase (FabZ)* , 2003, Journal of Biological Chemistry.

[14]  M. Blaser,et al.  Helicobacter pylori infection, a paradigm for chronic mucosal inflammation: pathogenesis and implications for eradication and prevention. , 1996, Advances in internal medicine.

[15]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[16]  Stanley Falkow,et al.  Global Transposon Mutagenesis and Essential Gene Analysis of Helicobacter pylori , 2004, Journal of bacteriology.

[17]  Jun Wang,et al.  Platensimycin is a selective FabF inhibitor with potent antibiotic properties , 2006, Nature.

[18]  P. Vagelos,et al.  The isolation and general properties of Escherichia coli malonyl coenzyme A-acyl carrier protein transacylase. , 1973, The Journal of biological chemistry.

[19]  L. Silver.  Multi-targeting by monotherapeutic antibacterials , 2007, Nature Reviews Drug Discovery.

[20]  J. Ganguly.  Studies on the mechanism of fatty acid synthesis. VII. Biosynthesis of fatty acids from malonyl CoA. , 1960, Biochimica et biophysica acta.

[21]  Kaixian Chen,et al.  Structural Basis for Catalytic and Inhibitory Mechanisms of β-Hydroxyacyl-acyl Carrier Protein Dehydratase (FabZ)* , 2008, Journal of Biological Chemistry.

[22]  Hualiang Jiang,et al.  A new beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from Helicobacter pylori: Molecular cloning, enzymatic characterization, and structural modeling. , 2005, Biochemical and biophysical research communications.

[23]  A. Schnapp,et al.  Phosphorylation-dependent proline isomerization catalyzed by Pin1 is essential for tumor cell survival and entry into mitosis. , 2000, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[24]  G. Fischer,et al.  Selective inactivation of parvulin-like peptidyl-prolyl cis/trans isomerases by juglone. , 1998, Biochemistry.

[25]  K. Soda.  Microbial sulfur amino acids: an overview. , 1987, Methods in enzymology.

[26]  M. Ljungman,et al.  The natural toxin juglone causes degradation of p53 and induces rapid H2AX phosphorylation and cell death in human fibroblasts. , 2005, Toxicology and applied pharmacology.

[27]  A. Greenleaf,et al.  Juglone, an inhibitor of the peptidyl-prolyl isomerase Pin1, also directly blocks transcription. , 2001, Nucleic acids research.

[28]  K. Nagata,et al.  Idebenone Acts against Growth of Helicobacter pylori by Inhibiting Its Respiration , 2006, Antimicrobial Agents and Chemotherapy.

[29]  R. Huber,et al.  Crystal structure of Escherichia coli cystathionine gamma-synthase at 1.5 A resolution. , 1998, The EMBO journal.

[30]  Jie J. Zheng,et al.  The structural biology of type II fatty acid biosynthesis. , 2005, Annual review of biochemistry.

[31]  C. Rock,et al.  Regulation of fatty acid biosynthesis in Escherichia coli. , 1993, Microbiological reviews.

[32]  R. Huber,et al.  Cloning, purification, crystallization, and preliminary X‐ray diffraction analysis of cystathionine γ‐synthase from E. coli , 1997, FEBS letters.

[33]  Aditi Gupta,et al.  Functional characterization of beta-ketoacyl-ACP reductase (FabG) from Plasmodium falciparum. , 2003, Biochemical and biophysical research communications.

[34]  R. Huber,et al.  Crystal structure of Escherichia coli cystathionine γ‐synthase at 1.5 Å resolution , 1998 .

[35]  C. Chignell,et al.  Cytotoxic action of juglone and plumbagin: a mechanistic study using HaCaT keratinocytes. , 2004, Chemical research in toxicology.

[36]  Deniz Tasdemir,et al.  Inhibition of Plasmodium falciparum fatty acid biosynthesis: evaluation of FabG, FabZ, and FabI as drug targets for flavonoids. , 2006, Journal of medicinal chemistry.

[37]  J. W. Campbell,et al.  Bacterial fatty acid biosynthesis: targets for antibacterial drug discovery. , 2001, Annual review of microbiology.

[38]  R. Heath,et al.  Roles of the FabA and FabZ β-Hydroxyacyl-Acyl Carrier Protein Dehydratases in Escherichia coli Fatty Acid Biosynthesis* , 1996, The Journal of Biological Chemistry.

[39]  Christian Drosten,et al.  Cinanserin Is an Inhibitor of the 3C-Like Proteinase of Severe Acute Respiratory Syndrome Coronavirus and Strongly Reduces Virus Replication In Vitro , 2005, Journal of Virology.

[40]  S. Williams,et al.  Helicobacter pylori: antibiotic resistance and eradication rates in Suffolk, UK, 1991-2001. , 2004, Journal of medical microbiology.

[41]  C. Raetz,et al.  An Escherichia coli gene (FabZ) encoding (3R)-hydroxymyristoyl acyl carrier protein dehydrase. Relation to fabA and suppression of mutations in lipid A biosynthesis. , 1994, The Journal of biological chemistry.

[42]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.