The catalytic activity of several COX II mutants assayed in the presence or absence of myricetin.

<p>The catalytic activity of COX II enzyme was based on measuring the formation of PGF_2α+PGE₂+PGD₂ from [¹⁴C]AA as substrate.</p>a<p>The number in parenthesis represents the % of increase or decrease over the corresponding control (in the absence of myricetin).</p>b<p>N.D., not detected.</p

The computed binding energy values (Δ<i>E</i>_binding) for the molecular docking study for the binding of myricetin, quercetin, chrysin or flavone with human COX I or COX II.

<p>The ligand-enzyme interaction energy value (Δ<i>E</i>_binding) was calculated using the following equation: Δ<i>E</i>_binding = <i>E</i>_complex−(<i>E</i>_COX+<i>E</i>_ligand), where <i>E</i>_complex was the potential energy for the complex of COX bound with the ligand, <i>E</i>_COX was the potential energy of the enzyme alone, and <i>E</i>_ligand was the potential energy for the ligand alone.</p

Identification of the binding sites for COX I and II by molecular docking approach.

<p><b>A</b>. The superimposed structures of COX I and II in complex with hematin and AA. The white labels indicate the two potential binding sites for quercetin as identified by the <i>Active-Site-Search</i> function in <i>InsightII</i>. <b>B</b>. Docking results for quercetin in Site-2 of COX I. <b>C</b>. Docking results for quercetin in Site-2 of COX II. <b>D</b>. Enlarged view of the interaction of quercetin with hematin and key amino acid residues in the peroxidase active sites of COX I. <b>E</b>. Enlarged view of the interaction of quercetin with hematin and key amino acid residues in the peroxidase active site of COX II. The protein structure was shown in the ribbon format in <b>A</b>, <b>B</b> and <b>C</b>. COX I was colored in pink and COX II in purple. AA was colored in light green for COX I and dark green for COX II. Quercetin was colored in light red for COX I and magenta for COX II. Carbon atoms in hematin were colored in yellow for COX I and orange for COX II whereas nitrogen atoms were colored in blue, oxygen atoms in red, and magnesium in silver. The green dashes represent the hydrogen bonds. Hematin, AA, key amino acid residues, and quercetin are shown in the ball and stick format. For amino acid residues, oxygen atoms are shown in red, carbon atoms in gray, and nitrogen atoms in blue. Hydrogens are omitted in these molecules.</p

Schematic depiction of the catalysis and inactivation mechanism of COX enzymes and their interaction with bioflavonoids.

<p>PPIX is for protoporphorin IX. Quercetin structure is shown as a representative bioactive bioflavonoid. Events in the peroxidase cycle are labeled with numbers to denote the sequence of occurrence.</p

Chemical structures of the bioflavonoids used in this study.

<p>The structure of flavone is enlarged to show the numbering of different carbon positions.</p

Primers used in the site-directed mutagenesis study.

<p>The sequences that are changed for each of the mutant COX proteins developed in this study are marked with underlines.</p

The 3-D QSAR/CoMFA color contour maps for COX I (A) and COX II (B).

<p>Note that quercetin was shown in the ball and stick format inside the field for demonstration. Oxygen, carbon, and hydrogen atoms are colored in red, gray, and blue, respectively. The contours of the steric maps are shown in yellow and green, and those of the electrostatic maps are shown in red and blue. Green contours indicate regions where a relatively bulkier substitution would increase the COX activity, whereas the yellow contours indicate areas where a bulkier substituent would decrease the COX activity. The red contours are regions where a negative-charged substitution likely would increase the COX activity whereas the blue contours showed areas where a negative-charged substitution would decrease the COX activity. Bioflavonoids with a higher ability to activate the COX enzymes are correlated with: (<b><i>i</i></b>) more bulkier substitute near green; (<b><i>ii</i></b>) less bulkier substitute near yellow; (<b><i>iii</i></b>) less negative charge near blue; and/or (<b><i>iv</i></b>) more negative charge near red. The figure in the lower panel shows the 90° rotation around the <i>x</i>-axis of the figure shown in the upper panel.</p

The 3-D QSAR/CoMFA analysis showing the correlation between the experimentally-determined COX-stimulating activity values and the predicted values.

<p>Nine representative bioflavonoids plus flavone were used in the analysis. The experimental values were based on measuring PGE₂ production, which was determined in our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012316#pone.0012316-Bai1" target="_blank">[15]</a>. Statistical parameters (<i>q</i>², <i>r</i>², PC, SEE, and F) for the CoMFA models of COX I and II are also listed in the figure.</p

Myricetin stimulates the catalytic activity of COX I and II (with or without aspirin pretreatment) when [¹⁴C]AA or PGG₂ is used as substrate.

<p>The incubation mixtures consisted of 20 µM [¹⁴C]AA (0.2 µCi) or 10 µM PGG₂ as substrate, COX I or COX II as enzyme (0.5 or 0.97 µg/mL, respectively), 10 mM EDTA, 1 mM reduced glutathione, 1 µM hematin, and myricetin in 200 µL Tris-HCl buffer (100 mM, pH 7.4). The reaction was incubated at 37°C for 5 min and terminated by adding 15 µL of 0.5 N HCl to each test tube. Ethyl acetate (600 µL) was added immediately for extraction. The dried extracts were re-dissolved in acetonitrile or EIA buffer (Cayman Co. Michigan, USA), and the metabolites were analyzed using HPLC (with radioactivity detection) when [¹⁴C]AA was used as substrate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012316#pone.0012316-Bai1" target="_blank">[15]</a> or using an EIA kit when PGG₂ was used as substrate. Note that in this experiment, the COX I and II enzymes with or without aspirin pretreatment were both tested. For aspirin pretreatment, enzymes were pre-incubated with aspirin at 0.5 mM for COX I or 5 mM for COX II for 30 min at room temperature and then were immediately used as the enzyme source in the assay.</p

Biochemical and Molecular Modeling Studies of the <i>O</i>-Methylation of Various Endogenous and Exogenous Catechol Substrates Catalyzed by Recombinant Human Soluble and Membrane-Bound Catechol-<i>O</i>-Methyltransferases

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) catalyzes the O-methylation of a wide array of catechol-containing substrates using s-adenosyl-l-methionine as the methyl donor. In the present study, we have cloned and expressed the human soluble and membrane-bound COMTs (S-COMT and MB-COMT, respectively) in Escherichia coli and have studied their biochemical characteristics for the O-methylation of representative classes of endogenous catechol substrates (catecholamines and catechol estrogens) as well as exogenous catechol substrates (bioflavonoids and tea catechins). Enzyme kinetic analyses showed that these two recombinant human COMTs are functionally active, with catalytic and kinetic properties nearly identical to those of crude or purified enzymes prepared from human tissues or cells. Kinetic parameters for the O-methylation of various substrates were characterized. In addition, computational modeling studies were conducted to better understand the molecular mechanisms for the different catalytic behaviors of human S- and MB-COMTs with respect to s-adenosyl-l-methionine, various substrates, and also the regioselectivity for the formation of mono-methyl ether products. Our modeling data showed that the binding energy values (ΔG) calculated for most substrates agreed well with the measured kinetic parameters. Also, our modeling data precisely predicted the regioselectivity for the O-methylation of these substrates at different hydroxyl groups, the predicted values matched nearly perfectly with the experimental data