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Structural basis for certain naturally occurring bioflavonoids to function as reducing co-substrates of cyclooxygenase I and II.

Wang P, Bai HW, Zhu BT - PLoS ONE (2010)

Bottom Line: The docking results were verified by biochemical analysis, which reveals that when the cyclooxygenase activity of COXs is inhibited by covalent modification, myricetin can still stimulate the conversion of PGG(2) to PGE(2), a reaction selectively catalyzed by the peroxidase activity.Using the site-directed mutagenesis analysis, we confirmed that Q189 at the peroxidase site of COX II is essential for bioflavonoids to bind and re-activate its catalytic activity.These findings provide the structural basis for bioflavonoids to function as high-affinity reducing co-substrates of COXs through binding to the peroxidase active site, facilitating electron transfer and enzyme re-activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Toxicology and Therapeutics, School of Medicine, University of Kansas Medical Center, Kansas City, Kansas, United States of America.

ABSTRACT

Background: Recent studies showed that some of the dietary bioflavonoids can strongly stimulate the catalytic activity of cyclooxygenase (COX) I and II in vitro and in vivo, presumably by facilitating enzyme re-activation. In this study, we sought to understand the structural basis of COX activation by these dietary compounds.

Methodology/principal findings: A combination of molecular modeling studies, biochemical analysis and site-directed mutagenesis assay was used as research tools. Three-dimensional quantitative structure-activity relationship analysis (QSAR/CoMFA) predicted that the ability of bioflavonoids to activate COX I and II depends heavily on their B-ring structure, a moiety known to be associated with strong antioxidant ability. Using the homology modeling and docking approaches, we identified the peroxidase active site of COX I and II as the binding site for bioflavonoids. Upon binding to this site, bioflavonoid can directly interact with hematin of the COX enzyme and facilitate the electron transfer from bioflavonoid to hematin. The docking results were verified by biochemical analysis, which reveals that when the cyclooxygenase activity of COXs is inhibited by covalent modification, myricetin can still stimulate the conversion of PGG(2) to PGE(2), a reaction selectively catalyzed by the peroxidase activity. Using the site-directed mutagenesis analysis, we confirmed that Q189 at the peroxidase site of COX II is essential for bioflavonoids to bind and re-activate its catalytic activity.

Conclusions/significance: These findings provide the structural basis for bioflavonoids to function as high-affinity reducing co-substrates of COXs through binding to the peroxidase active site, facilitating electron transfer and enzyme re-activation.

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Myricetin stimulates the catalytic activity of COX I and II (with or without aspirin pretreatment) when [14C]AA or PGG2 is used as substrate.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 [15] 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.
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pone-0012316-g005: Myricetin stimulates the catalytic activity of COX I and II (with or without aspirin pretreatment) when [14C]AA or PGG2 is used as substrate.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 [15] 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.

Mentions: To provide experimental evidence for the computational model that bioflavonoids can stimulate the catalytic activity of COXs by binding to the peroxidase site but not the cyclooxygenase site, we studied the stimulatory effect of myricetin (a representative bioflavonoid) on the catalytic activity of COX I and II pretreated with aspirin, which can covalently acetylate and thereby inactivate the cyclooxygenase active site in these enzymes. To selectively test the effect of myricetin on the peroxidase activity of COX enzymes, PGG2 was used as a substrate to bypass the cyclooxygenation step (i.e., the conversion of AA to PGG2). As shown in Figure 5A and 5D, pretreatment of COX I and II with aspirin (0.5 and 5 mM, respectively) strongly inhibited the cyclooxygenase activity by 72% and 70%, respectively, when [14C]AA was used as substrate. However, when PGG2 was used as substrate, aspirin-pretreated COX I and II did not exert the same level of inhibition of their COX activity, which was as expected.


Structural basis for certain naturally occurring bioflavonoids to function as reducing co-substrates of cyclooxygenase I and II.

Wang P, Bai HW, Zhu BT - PLoS ONE (2010)

Myricetin stimulates the catalytic activity of COX I and II (with or without aspirin pretreatment) when [14C]AA or PGG2 is used as substrate.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 [15] 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2925883&req=5

pone-0012316-g005: Myricetin stimulates the catalytic activity of COX I and II (with or without aspirin pretreatment) when [14C]AA or PGG2 is used as substrate.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 [15] 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.
Mentions: To provide experimental evidence for the computational model that bioflavonoids can stimulate the catalytic activity of COXs by binding to the peroxidase site but not the cyclooxygenase site, we studied the stimulatory effect of myricetin (a representative bioflavonoid) on the catalytic activity of COX I and II pretreated with aspirin, which can covalently acetylate and thereby inactivate the cyclooxygenase active site in these enzymes. To selectively test the effect of myricetin on the peroxidase activity of COX enzymes, PGG2 was used as a substrate to bypass the cyclooxygenation step (i.e., the conversion of AA to PGG2). As shown in Figure 5A and 5D, pretreatment of COX I and II with aspirin (0.5 and 5 mM, respectively) strongly inhibited the cyclooxygenase activity by 72% and 70%, respectively, when [14C]AA was used as substrate. However, when PGG2 was used as substrate, aspirin-pretreated COX I and II did not exert the same level of inhibition of their COX activity, which was as expected.

Bottom Line: The docking results were verified by biochemical analysis, which reveals that when the cyclooxygenase activity of COXs is inhibited by covalent modification, myricetin can still stimulate the conversion of PGG(2) to PGE(2), a reaction selectively catalyzed by the peroxidase activity.Using the site-directed mutagenesis analysis, we confirmed that Q189 at the peroxidase site of COX II is essential for bioflavonoids to bind and re-activate its catalytic activity.These findings provide the structural basis for bioflavonoids to function as high-affinity reducing co-substrates of COXs through binding to the peroxidase active site, facilitating electron transfer and enzyme re-activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Toxicology and Therapeutics, School of Medicine, University of Kansas Medical Center, Kansas City, Kansas, United States of America.

ABSTRACT

Background: Recent studies showed that some of the dietary bioflavonoids can strongly stimulate the catalytic activity of cyclooxygenase (COX) I and II in vitro and in vivo, presumably by facilitating enzyme re-activation. In this study, we sought to understand the structural basis of COX activation by these dietary compounds.

Methodology/principal findings: A combination of molecular modeling studies, biochemical analysis and site-directed mutagenesis assay was used as research tools. Three-dimensional quantitative structure-activity relationship analysis (QSAR/CoMFA) predicted that the ability of bioflavonoids to activate COX I and II depends heavily on their B-ring structure, a moiety known to be associated with strong antioxidant ability. Using the homology modeling and docking approaches, we identified the peroxidase active site of COX I and II as the binding site for bioflavonoids. Upon binding to this site, bioflavonoid can directly interact with hematin of the COX enzyme and facilitate the electron transfer from bioflavonoid to hematin. The docking results were verified by biochemical analysis, which reveals that when the cyclooxygenase activity of COXs is inhibited by covalent modification, myricetin can still stimulate the conversion of PGG(2) to PGE(2), a reaction selectively catalyzed by the peroxidase activity. Using the site-directed mutagenesis analysis, we confirmed that Q189 at the peroxidase site of COX II is essential for bioflavonoids to bind and re-activate its catalytic activity.

Conclusions/significance: These findings provide the structural basis for bioflavonoids to function as high-affinity reducing co-substrates of COXs through binding to the peroxidase active site, facilitating electron transfer and enzyme re-activation.

Show MeSH