Structural basis for catalytic and inhibitory mechanisms of beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ)

beta-Hydroxyacyl-acyl carrier protein dehydratase (FabZ) is an important enzyme for the elongation cycles of both saturated and unsaturated fatty acids biosyntheses in the type II fatty acid biosynthesis system (FAS II) pathway. FabZ has been an essential target for the discovery of compounds effective against pathogenic microbes. In this work, to characterize the catalytic and inhibitory mechanisms of FabZ, the crystal structures of the FabZ of Helicobacter pylori (HpFabZ) and its complexes with two newly discovered inhibitors have been solved. Different from the structures of other bacterial FabZs, HpFabZ contains an extra short two-turn alpha-helix (alpha4) between alpha3 and beta3, which plays an important role in shaping the substrate-binding tunnel. Residue Tyr-100 at the entrance of the tunnel adopts either an open or closed conformation in the crystal structure. The crystal structural characterization, the binding affinity determination, and the enzymatic activity assay of the HpFabZ mutant (Y100A) confirm the importance of Tyr-100 in catalytic activity and substrate binding. Residue Phe-83 at the exit tunnel was also refined in two alternative conformations, leading the tunnel to form an L-shape and U-shape. All these data thus contributed much to understanding the catalytic mechanism of HpFabZ. In addition, the co-crystal structures of HpFabZ with its inhibitors have suggested that the enzymatic activity of HpFabZ could be inhibited either by occupying the entrance of the tunnel or plugging the tunnel to prevent the substrate from accessing the active site. Our study has provided some insights into the catalytic and inhibitory mechanisms of FabZ, thus facilitating antibacterial agent development.     

苦苣菜属5种植物的黄酮类成分含量分析

菊科（Compositae）苦苣菜属（Sonchus L．）的许多种类在民间作为药用植物使用,大多具有清热解毒、消肿排脓及凉血利湿等功效。苦苣菜属植物主要含有黄酮类及倍半萜类活性成分,其中苦苣菜（S．oleraceus L．）中的总黄酮对脑缺血缺氧小鼠及实验性肝损伤均有明显的保护作用。     

A new beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from Helicobacter pylori: Molecular cloning, enzymatic characterization, and structural modeling

Helicobacter pylori is a gram-negative pathogenic bacterium that causes peptic ulcer disease and gastric cancer, and studies of the related potent enzymes associated with this bacterium are urgent for the discovery of novel drug targets. In bacteria, beta-hydroxyacyl-acyl carrier protein (ACP) dehydratase (FabZ) is a potent enzyme in fatty acid biosynthesis and catalyzes the dehydration of beta-hydroxyacyl-ACP to trans-2-acyl-ACP. In this study, the cloning and enzymatic characterization of FabZ from H. pylori strain SS1 (HpFabZ) were reported, and the gene sequence of HpfabZ was deposited in the GenBank database. Enzyme dynamic analysis showed that HpFabZ had a K(m) of 82.6+/-4.3 microM toward its substrate analog crotonoyl-CoA. Dynamic light scattering and native-PAGE investigations suggested that HpFabZ exists as hexamer in native state. Enzymatic characterization and thermal-induced unfolding analysis based on circular dichroism spectral measurements indicated that HpFabZ is very stable against high temperature (90 degrees C). Such a high stability of HpFabZ was well elucidated by the strong H-bonds and hydrophobic interactions among the HpFabZ hexamer as investigated in the modeled HpFabZ hexamer structure. Our current study is hoped to provide useful information in better understanding the FabZ of H. pylori strain and further supply possible hints in the discovery of anti-bacterial compounds using HpFabZ as target.     

Characterization of quercetin binding site on DNA gyrase

Gyrases are DNA topology modifying enzymes present only in prokaryotes which makes them an attractive target for antibacterial drugs. Quercetin, one of the most abundant natural flavonoids, inhibits supercoiling activity of bacterial gyrase and induces DNA cleavage. It has been generally assumed that the mechanism of flavonoid inhibition is based on interaction with DNA. We show that quercetin binds to the 24 kDa fragment of gyrase B of Escherichia coli with a K(D) value of 15 microM and inhibits ATPase activity of gyrase B. Its binding site overlaps with ATP binding pocket and could be competitively replaced by either ATP or novobiocin. The structural model of quercetin-gyrase complex was prepared, based on the close similarity with ATP and quercetin binding sites of the src family tyrosine kinase Hck. We propose that quercetin inhibits gyrases through two different mechanisms based either on interaction with DNA or with ATP binding site of gyrase.     

Influence of dietary flavonoids on the glycation of plasma proteins

It has been suggested that the increasing glycation in diabetes can influence the ability of plasma proteins to bind to small molecules. Herein, the influence of flavonoids on the glycation of plasma proteins was investigated. After being incubated with glucose at 37 °C, the levels of glycated albumin (HGA) were significantly improved in healthy human plasma proteins (HPP). The inhibitory effects of flavonoids against the formation of advanced glycation products (AGEs) in HPP were determined as: galangin > apigenin > kaempferol ≈ luteolin > myricetin > quercetin. After being combined with 20 μmol L?1 of quercetin for 11 days, the fresh plasma with δ-glucose caused 323.05-32.07% inhibition of HGA formation in type II diabetes plasma proteins (TPP). Luteolin showed weak inhibition of HGA formation in TPP. However, kaempferol, galangin and apigenin hardly inhibited the formation of HGA in TPP. These results showed that more hydroxyl groups on ring B of flavonoids will enhance the inhibitory effects on the HGA formation in TPP.     

Comparative study of the binding of 3 flavonoids to the fat mass and obesity‐associated protein by spectroscopy and molecular modeling

This study aims to investigate the interaction between 3 flavonoids (quercetin, apigenin, and naringenin) and fat mass and obesity‐associated protein by fluorescence, ultraviolet‐visible absorption spectroscopy, and molecular modeling. Results indicate that the intrinsic fluorescence of fat mass and obesity‐associated protein can be quenched by the 3 flavonoids through a static quenching procedure. Thermodynamic analysis and molecular modeling results suggest that hydrophobic interaction and hydrogen bond forces play the major roles in the binding process. Moreover, results also show that the rank order of quenching constant and binding constant is quercetin > apigenin > naringenin.     

Molecular modeling of flavonoids that inhibits xanthine oxidase

The inhibition of xanthine oxidase activity by various flavonoids was assessed. All of the tested flavonoids were competitive inhibitors, and from the kinetic analysis suggested that flavonoids bind to the reactive site. To further understand the stereochemistry between these flavonoids and xanthine oxidase, structure-based molecular modeling was performed. Apigenin was the most potent inhibitor which showed the most favorable interaction in the reactive site. The bicyclic benzopyranone ring of apigenin stacked with phenyl of Phe 914, and the phenolic group stretched to the space surrounding with several hydrophobic residues. Quercetin and myricetin composed a 3-hydroxyl group on benzopyranone which resulting in reduction of binding affinity. The phenolic group of genistein positioned in opposite orientation comparison with apigenin, and resulted in a weaker interaction with xanthine oxidase. Isovitexin showed the weakest inhibitory effect among the compounds tested. The bulky group of sugar in isovitexin may hamper its interaction with xanthine oxidase.     

Cyclohexanoid protoflavanones from the stem-bark and roots of Ongokea gore

Phytochemical investigation of root and stem-bark of the West African medicinal plant Ongokea gore resulted in the isolation of four novel flavonoids with an unusual cyclohexyl substituent instead of the common aromatic ring B. The structures of the isolated compounds were elucidated by spectroscopic methods, mainly 1D and 2D NMR, and subsequently, the structures were corroborated by chemical conversion to (-)-(S)-sakuranetin. The absolute configurations, and preferred conformations were determined by NOE experiments and CD measurements.     

Complex I and cytochrome c are molecular targets of flavonoids that inhibit hydrogen peroxide production by mitochondria

Flavonoids can protect cells from different insults that lead to mitochondria-mediated cell death, and epidemiological data show that some of these compounds attenuate the progression of diseases associated with oxidative stress and mitochondrial dysfunction. In this work, a screening of 5 flavonoids representing major subclasses showed that they display different effects on H?O? production by mitochondria isolated from rat brain and heart. Quercetin, kaempferol and epicatechin are potent inhibitors of H?O? production by mitochondria from both tissues (IC?? approximately 1-2 μM), even when H?O? production rate was stimulated by the mitochondrial inhibitors rotenone and antimycin A. Although the rate of oxygen consumption was unaffected by concentrations up to 10 μM of these flavonoids, quercetin, kaempferol and apigenin inhibited complex I activity, while up to 100 μM epicatechin produced less than 20% inhibition. The extent of this inhibition was found to be dependent on the concentration of coenzyme Q in the medium, suggesting competition between the flavonoids and ubiquinone for close binding sites in the complex. In contrast, these flavonoids did not significantly inhibit the activity of complexes II and III, and did not affect the redox state of complex IV. However, we have found that epicatechin, quercetin and kaempferol are able to stoichiometrically reduce purified cytochrome c. Our results reveal that mitochondria are a plausible main target of flavonoids mediating, at least in part, their reported preventive actions against oxidative stress and mitochondrial dysfunction-associated pathologies.     

Antibacterial activity of pure flavonoids isolated from mosses

Seven pure flavonoids were isolated and identified from five moss species. The flavonoids were the flavones apigenin, apigenin-7-O-triglycoside, lucenin-2, luteolin-7-O-neohesperidoside, saponarine and vitexin; and the biflavonoid bartramiaflavone. Some of these flavonoids were shown to have pronounced antibacterial effects against Enterobacter cloaceae, E. aerogenes and Pseudomonas aeruginosa (minimal bacteriostatic concentration MIC in the range of 4-2048 micrograms/ml). Because of their antibacterial spectrum mainly active against Gram negative bacterial strains, responsible for severe opportunistic infections and resistant to common antibacterial therapy, these flavonoids may be important tools in antibacterial strategies.     

Bulky aromatic amino acids increase the antibacterial activity of 15-residue bovine lactoferricin derivatives.

A model peptide, FKCRRWQWRMKKLGA, residues 17-31 of bovine lactoferricin, has been subjected to structure-antibacterial activity relationship studies. The two Trp residues are very important for antibacterial activity, and analogue studies have demonstrated the significance of the size, shape and aromatic character of the side chains. In the current study we have replaced Trp residues in the model peptide with bulky aromatic amino acids to elucidate further the importance of size and shape. The counterproductive Cys residue in position 3 was also replaced by these aromatic amino acids. The largest aromatic amino acids employed resulted in the most active peptides. The peptides containing these hydrophobic residues were generally more active against Staphylococcus aureus than against Escherichia coli, indicating that the bacterial specificity as well as the antibacterial efficiency can be altered by employing large hydrophobic aromatic amino acid residues.     

Rutin-enhanced antibacterial activities of flavonoids against Bacillus cereus and Salmonella enteritidis

The antibacterial activities of flavonoids were found by the paper disk method to be enhanced by combining or mixing them. The combinations of quercetin and quercitrin, quercetin and morin, and quercetin and rutin were much more active than either flavonoid alone. Although rutin did not show activity in itself, the antibacterial activities of quercetin and morin were enhanced in the presence of rutin. The antibacterial activities of flavonoids, in combination with morin and rutin, were evaluated, based on the minimum inhibition concentration (MIC) in a liquid culture, by using Salmonella enteritidis and Bacillus cereus as the test bacteria. The activities of galangin, kaempherol, myricetin and fisetin were each enhanced in the presence of rutin when S. enteritidis was used as the test bacterium. The MIC value for kaempherol was markedly decreased by the addition of rutin. Morin inhibited DNA synthesis, and this effect was promoted by rutin at a concentration of 25 microg/ml.     

Insect immunity: isolation and structure of cecropins B and D from pupae of the Chinese oak silk moth, Antheraea pernyi

The immune system in the Chinese oak silk moth, Antheraea pernyi, has been compared with that of the Cecropia moth which has been characterized earlier. Antibacterial activity against Escherichia coli was induced in diapausing pupae by injection of viable E. coli or Enterobacter cloacae. The activity reached a maximum on day 7-8 after which the response gradually declined. The pupae produced a set of immune proteins with P4 and P5 as major labelled components similar to that earlier found in Cecropia. The major antibacterial factor in A. pernyi was cecropin D. A procedure is described for the isolation of cecropin B and D, which is in principle similar to the one used for the isolation of the corresponding cecropins from Cecropia pupae. Amino acid sequence analyses of the A. pernyi cecropins show the D form to contain 36 amino acid residues and that both cecropins have blocked C-termini. The general structure of cecropins having a charged N-terminal region (residues 1-21) followed by a long hydrophobic stretch (residues 22-32) is well conserved. Cecropin B and D from A. pernyi differ from the corresponding proteins in Cecropia by four and three conservative amino acid replacements, respectively. The homology between the cecropins from the two insects suggests that they orginate from a single ancestral gene. The antibacterial activity was tested against nine different bacterial species. Evolutionary aspects of the cecropins are discussed.     

Antibacterial and free-radical scavenging activities of Sonoran propolis

AIMS: To evaluate the antibacterial and free-radical scavenging (FRS) activities of propolis collected from three different areas of Sonoran Desert in northwestern Mexico [Pueblo de Alamos (PAP), Ures (UP) and Caborca (CP)]. METHODS AND RESULTS: The antibacterial and FRS activities of Sonoran propolis were determined by the broth microdilution method and the DPPH (1,1-diphenyl-2-picrylhydracyl) assay, respectively. Propolis samples had antibacterial activity against only Gram-positive bacteria. The UP sample showed the highest antibacterial activity against Staphylococcus aureus [minimal inhibitory concentration (MIC) 100 microg ml(-1)] in a concentration-dependent manner (UP > CP > PAP). Caffeic acid phenethyl ester (CAPE), a UP propolis constituent, had very high growth-inhibitory activity towards Gram-positive bacteria, particularly against S. aureus (MIC 0.1 mmol l(-1)). To our knowledge, this is the first study showing a strong antibacterial activity of CAPE against S. aureus. Additionally, propolis CP exhibited high FRS activity (86% +/- 0.3 at 100 microg ml(-1)) comparable with those of the reference antioxidants vitamin C (87.4% +/- 1.7 at 70 micromol l(-1)) and BHT (66.07% +/- 0.76 at 140 micromol l(-1)). The propolis compounds CAPE and rutin showed high FRS activity (90.4% +/- 0.2 and 88.5% +/- 0.8 at 70 micromol l(-1), respectively). CONCLUSIONS: Sonoran propolis UP and CAPE had strong antibacterial activity against S. aureus. In addition, propolis CP showed potent FRS activity comparable with those of vitamin C and BHT. SIGNIFICANCE AND IMPACT OF THE STUDY: The strong antibacterial and antioxidant properties of Sonoran propolis and some of its constituents support further studies on the clinical applications of this natural bee product against S. aureus and several oxidative damage-related diseases.     

Interactions between flavonoids and hemoglobin in lecithin liposomes

In this paper, the binding of flavonoids (quercetin and rutin) to hemoglobin (Hb) have been investigated by fluorescence, absorption spectroscopy and circular dichroism (CD) spectroscopy. The binding parameters and binding mode between flavonoids and Hb are determined and the results of CD and synchronous fluorescence spectra indicate a conformational change of Hb with addition of flavonoids. The effects of lecithin liposomes on the binding parameter of quercetin and rutin to Hb are also studied. When incorporated into liposome, flavonoids can reduce the fluorescence of tryptophanyl residues of Hb to a lesser extent. The difference of the structure characteristics between quercetin and rutin has a significant effect on their binding affinity for Hb.     

Polyphenolic compounds of a new biologically active extract from immortelle sandy flowers (<Emphasis Type="Italic">Helichrysum arenarium</Emphasis> (L.) Moench.)

The chemical composition of a new bioactive extract from immortelle sandy (Helichrysum arenarium (L.) Moench.) was investigated. Naringin, its soluble aggregate, prunin, quercetin, apigenin, naringenin, apigenin 5-O-glucoside, and isosalipurposide were found among the flavonoids. The tested immortelle sandy extract was shown to contain known dimers, trimers, or more complex aggregates. The compounds had similar spectral but substantially different chromatographic characteristics, which can be used for their identification based on the corresponding data on flavonoids glycosylated at different positions and/or carbohydrates of various complexities, which are also characterized by similar absorption spectra and different retention times. It was found by molecular absorption spectroscopy that the immortelle extract contained 73.48 mg of flavonoids relative to rutin or 17.94 mg relative to quercetin per 1 g of dry extract weight, which corresponded to 20.99 and 5.13%, respectively. The immortelle extract obtained by the method proposed displayed antitumor activity against the transplanted sarcoma 45 and had a beneficial effect on animals in general.     

Isolation and studies of the mutagenic activity in the Ames test of flavonoids naturally occurring in medical herbs

Quercetin, rhamnetin, isorhamnetin, apigenin and luteolin were isolated from medicinal herbs: Erigeron canadensis L., Anthyllis vulneraria L. and Pyrola chloranta L. The mutagenicity of these naturally occurring flavonoids was tested by the Ames method with S. typhimurium strains TA1535, TA1538, TA97, TA98, TA100 and TA102 in the presence and absence of metabolic activation. Of the above flavonoids only quercetin and rhamnetin revealed mutagenic activity in the Ames test. Quercetin induced point mutations in strains TA97, TA98, TA100 and TA102 of S. typhimurium. The presence of S9 rat liver microsome fraction markedly enhanced the mutagenic activity of quercetin in these strains. Rhamnetin appeared to be a much weaker mutagen in the Ames test. The compound induced mutations in strains TA97, TA98 and TA100 of S. typhimurium but only in the presence of metabolic activation.Comparison of the structure of the studied flavonoids with their mutagenic activity indicates that the mutagenicity of flavonoids is dependent on the presence of hydroxyl groups in the 3′ and 4′ positions of the B ring, and that the presence of a free hydroxy or methoxy group in the 7 position of the A ring also probably contributes to the appearance of mutagenic activity of flavonoids in the Ames test. It also appeared that the presence of methoxy groups, particularly in the B ring of the flavonoid molecule, markedly decreases the mutagenic activity of the compound.     

Flavonoids are potential inhibitors of glucose uptake in U937 cells.

Flavonoids are a group of polyphenolic compounds ubiquitously found in plants including fruits, and vegetables. Broad ranges of the biological activities of flavonoids have been reported using in vitro studies. I report that several natural flavonoids blocked glucose uptake in myelocytic U937 cells. Although there were some variations in the blocking activity of individual flavonoids, approximately half of the glucose uptake was blocked by flavonoids at the concentrations of 8-50 microM. The decreasing order of the blocking activity was fisetin >/= myricetin >/= quercetin >/= apigenin > genistein > cyanidin > daidzein >/= hesperetin > naringenin > catechin. Fisetin showed approximately 50% inhibition of glucose uptake at a concentration of 8 microM. Similar patterns of the inhibition were observed in lymphocytic Jurkat cells. Fisetin and quercetin inhibited glucose transport in a competitive manner. K(i) values for fisetin and quercetin were proximately 9 and 12 microM, respectively. This study showed that some types of natural flavonoids block glucose uptake in U937 cells and that natural flavonoids could be used as alternative blockers of glucose uptake in vitro.     

Peroxidative metabolism of apigenin and naringenin versus luteolin and quercetin: glutathione oxidation and conjugation

GSH was readily depleted by a flavonoid, H(2)O(2), and peroxidase mixture but the products formed were dependent on the redox potential of the flavonoid. Catalytic amounts of apigenin and naringenin but not kaempferol (flavonoids that contain a phenol B ring) when oxidized by H(2)O(2) and peroxidase co-oxidized GSH to GSSG via a thiyl radical which could be trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) to form a DMPO-glutathionyl radical adduct detected by ESR spectroscopy. On the other hand, quercetin and luteolin (flavonoids that contain a catechol B ring) or kaempferol depleted GSH stoichiometrically without forming a thiyl radical or GSSG. Quercetin, luteolin, and kaempferol formed mono-GSH and bis-GSH conjugates, whereas apigenin and naringenin did not form GSH conjugates. MS/MS electrospray spectroscopy showed that mono-GSH conjugates for quercetin and luteolin had peaks at m/z 608 [M + H](+) and m/z 592 [M + H](+) in the positive-ion mode, respectively. (1)H NMR spectroscopy showed that the GSH was bound to the quercetin A ring. Spectral studies indicated that at a physiological pH the luteolin-SG conjugate was formed from a product with a UV maximum absorbance at 260 nm that was reducible by potassium borohydride. The quercetin-SG conjugate or kaempferol-SG conjugate on the other hand was formed from a product with a UV maximum absorbance at 335 nm that was not reducible by potassium borohydride. These results suggest that GSH was oxidized by apigenin/naringenin phenoxyl radicals, whereas GSH conjugate formation involved the o-quinone metabolite of luteolin or the quinoid (quinone methide) product of quercetin/kaempferol.     

Elucidation of active site residues of Arabidopsis thaliana flavonol synthase provides a molecular platform for engineering flavonols

Arabidopsis thaliana flavonol synthase (aFLS) catalyzes the production of quercetin, which is known to possess multiple medicinal properties. aFLS is classified as a 2-oxoglutarate dependent dioxygenase as it requires ferrous iron and 2-oxoglutarate for catalysis. In this study, the putative residues for binding ferrous iron (H221, D223 and H277), 2-oxoglutarate (R287 and S289) and dihydroquercetin (H132, F134, K202, F293 and E295) were identified via computational analyses. To verify the proposed roles of the identified residues, 15 aFLS mutants were constructed and their activities were examined via a spectroscopic assay designed in this study. Mutations at H221, D223, H277 and R287 completely abolished enzymes activities, supporting their importance in binding ferrous iron and 2-oxoglutarate. However, mutations at the proposed substrate binding residues affected the enzyme catalysis differently such that the activities of K202 and F293 mutants drastically decreased to approximately 10% of the wild-type whereas the H132F mutant exhibited approximately 20% higher activity than the wild-type. Kinetic analyses established an improved substrate binding affinity in H132F mutant (Km: 0.027+/-0.0028 mM) compared to wild-type (Km: 0.059+/-0.0063 mM). These observations support the notion that aFLS can be selectively mutated to improve the catalytic activity of the enzyme for quercetin production.