CYP2U1, a Novel Human Thymus- and Brain-specific Cytochrome P450, Catalyzes - and ( -1)-Hydroxylation of Fatty Acids (original) (raw)
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The Journal of biological chemistry, 2004
Long chain fatty acids have recently emerged as critical signaling molecules in neuronal, cardiovascular, and renal processes, yet little is presently known about the precise mechanisms controlling their tissue distribution and bioactivation. We have identified a novel cytochrome P450, CYP2U1, which may play an important role in modulating the arachidonic acid signaling pathway. Northern blot and real-time PCR analysis demonstrated that CYP2U1 transcripts were most abundant in the thymus and the brain (cerebellum), indicating a specific physiological role for CYP2U1 in these tissues. Recombinant human CYP2U1 protein, expressed in baculovirus-infected Sf9 insect cells, was found to metabolize arachidonic acid exclusively to two region-specific products as determined by liquid chromatography-mass spectrometry. These metabolites were identified as 19- and 20-hydroxy-modified arachidonic acids by liquid chromatography-tandem mass spectrometry analysis. In addition to omega/omega-1 hydro...
Arachidonic Acid-metabolizing Cytochrome P450 Enzymes Are Targets of -3 Fatty Acids
Journal of Biological Chemistry, 2010
Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protect against cardiovascular disease by largely unknown mechanisms. We tested the hypothesis that EPA and DHA may compete with arachidonic acid (AA) for the conversion by cytochrome P450 (CYP) enzymes, resulting in the formation of alternative, physiologically active, metabolites. Renal and hepatic microsomes, as well as various CYP isoforms, displayed equal or elevated activities when metabolizing EPA or DHA instead of AA. CYP2C/2J isoforms converting AA to epoxyeicosatrienoic acids (EETs) preferentially epoxidized the-3 double bond and thereby produced 17,18-epoxyeicosatetraenoic (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) from EPA and DHA. We found that these-3 epoxides are highly active as antiarrhythmic agents, suppressing the Ca 2؉-induced increased rate of spontaneous beating of neonatal rat cardiomyocytes, at low nanomolar concentrations. CYP4A/4F isoforms-hydroxylating AA were less regioselective toward EPA and DHA, catalyzing predominantlyand minus 1 hydroxylation. Rats given dietary EPA/DHA supplementation exhibited substantial replacement of AA by EPA and DHA in membrane phospholipids in plasma, heart, kidney, liver, lung, and pancreas, with less pronounced changes in the brain. The changes in fatty acids were accompanied by concomitant changes in endogenous CYP metabolite profiles (e.g. altering the EET/EEQ/EDP ratio from 87:0:13 to 27🔞55 in the heart). These results demonstrate that CYP enzymes efficiently convert EPA and DHA to novel epoxy and hydroxy metabolites that could mediate some of the beneficial cardiovascular effects of dietary-3 fatty acids. Cytochrome P450 (CYP) 3 enzymes initiate the so-called "third branch" of the arachidonic acid (AA; 20:4n-6) cascade (1). Members of the CYP2C and CYP2J subfamilies function as * This work was supported in part by grants SCHU822/5 and FOR1054 from the Deutsche Forschungsgemeinschaft (to D. N. M. and W.-H. S.). The animal studies were supported by Solvay Arzneimittel GmbH.
Arachidonic Acid-metabolizing Cytochrome P450 Enzymes Are Targets of ω-3 Fatty Acids*
Journal of Biological Chemistry, 2010
Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protect against cardiovascular disease by largely unknown mechanisms. We tested the hypothesis that EPA and DHA may compete with arachidonic acid (AA) for the conversion by cytochrome P450 (CYP) enzymes, resulting in the formation of alternative, physiologically active, metabolites. Renal and hepatic microsomes, as well as various CYP isoforms, displayed equal or elevated activities when metabolizing EPA or DHA instead of AA. CYP2C/2J isoforms converting AA to epoxyeicosatrienoic acids (EETs) preferentially epoxidized the-3 double bond and thereby produced 17,18-epoxyeicosatetraenoic (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) from EPA and DHA. We found that these-3 epoxides are highly active as antiarrhythmic agents, suppressing the Ca 2؉-induced increased rate of spontaneous beating of neonatal rat cardiomyocytes, at low nanomolar concentrations. CYP4A/4F isoforms-hydroxylating AA were less regioselective toward EPA and DHA, catalyzing predominantlyand minus 1 hydroxylation. Rats given dietary EPA/DHA supplementation exhibited substantial replacement of AA by EPA and DHA in membrane phospholipids in plasma, heart, kidney, liver, lung, and pancreas, with less pronounced changes in the brain. The changes in fatty acids were accompanied by concomitant changes in endogenous CYP metabolite profiles (e.g. altering the EET/EEQ/EDP ratio from 87:0:13 to 27🔞55 in the heart). These results demonstrate that CYP enzymes efficiently convert EPA and DHA to novel epoxy and hydroxy metabolites that could mediate some of the beneficial cardiovascular effects of dietary-3 fatty acids. Cytochrome P450 (CYP) 3 enzymes initiate the so-called "third branch" of the arachidonic acid (AA; 20:4n-6) cascade (1). Members of the CYP2C and CYP2J subfamilies function as * This work was supported in part by grants SCHU822/5 and FOR1054 from the Deutsche Forschungsgemeinschaft (to D. N. M. and W.-H. S.). The animal studies were supported by Solvay Arzneimittel GmbH.
Arachidonic Acid-Metabolizing Cytochrome P450 Enzymes Are Targets of OMEGA3 Fatty Acids
Journal of Biological Chemistry, 2010
Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protect against cardiovascular disease by largely unknown mechanisms. We tested the hypothesis that EPA and DHA may compete with arachidonic acid (AA) for the conversion by cytochrome P450 (CYP) enzymes, resulting in the formation of alternative, physiologically active, metabolites. Renal and hepatic microsomes, as well as various CYP isoforms, displayed equal or elevated activities when metabolizing EPA or DHA instead of AA. CYP2C/2J isoforms converting AA to epoxyeicosatrienoic acids (EETs) preferentially epoxidized the-3 double bond and thereby produced 17,18-epoxyeicosatetraenoic (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) from EPA and DHA. We found that these-3 epoxides are highly active as antiarrhythmic agents, suppressing the Ca 2؉-induced increased rate of spontaneous beating of neonatal rat cardiomyocytes, at low nanomolar concentrations. CYP4A/4F isoforms-hydroxylating AA were less regioselective toward EPA and DHA, catalyzing predominantlyand minus 1 hydroxylation. Rats given dietary EPA/DHA supplementation exhibited substantial replacement of AA by EPA and DHA in membrane phospholipids in plasma, heart, kidney, liver, lung, and pancreas, with less pronounced changes in the brain. The changes in fatty acids were accompanied by concomitant changes in endogenous CYP metabolite profiles (e.g. altering the EET/EEQ/EDP ratio from 87:0:13 to 27🔞55 in the heart). These results demonstrate that CYP enzymes efficiently convert EPA and DHA to novel epoxy and hydroxy metabolites that could mediate some of the beneficial cardiovascular effects of dietary-3 fatty acids. Cytochrome P450 (CYP) 3 enzymes initiate the so-called "third branch" of the arachidonic acid (AA; 20:4n-6) cascade (1). Members of the CYP2C and CYP2J subfamilies function as * This work was supported in part by grants SCHU822/5 and FOR1054 from the Deutsche Forschungsgemeinschaft (to D. N. M. and W.-H. S.). The animal studies were supported by Solvay Arzneimittel GmbH.
Journal of Lipid Research, 2000
The demonstration of in vivo arachidonic acid epoxidation and-hydroxylation established the cytochrome P450 epoxygenase and /-1 hydroxylase as formal metabolic pathways and as members of the arachidonate metabolic cascade. The characterization of the potent biological activities associated with several of the cytochrome P450-derived eicosanoids suggested new and important functional roles for these enzymes in cellular, organ, and body physiology, including the control of vascular reactivity and systemic blood pressures. Past and current advances in cytochrome P450 biochemistry and molecular biology facilitate the characterization of cytochrome P450 isoforms responsible for tissue/organ specific arachidonic acid epoxidation and /-1 hydroxylation, and thus, the analysis of cDNA and/or gene specific functional phenotypes. The combined application of physiological, biochemical, molecular, and genetic approaches is beginning to provide new insights into the physiological and/or pathophysiological significance of these enzymes, their endogenous substrates, and products.-Capdevila,
Metabolism of eicosapentaenoic and docosahexaenoic acids by recombinant human cytochromes P450
Archives of Biochemistry and Biophysics, 2008
Epoxidation and hydroxylation of arachidonic acid (AA) are both catalyzed by cytochromes P450s (CYPs). The oxidized metabolites are known to be involved in the regulation of vascular tone and renal function. By using a panel of 15 human recombinant CYPs, this study demonstrates that other polyunsaturated long-chain fatty acids (PUFA-LC), especially the x3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are also epoxidised. The regioselectivity of epoxidation of four PUFA-LC by CYPs was investigated. Among the several CYPs tested, CYP2C9/2C19 and 1A2 were the most efficient in EPA and DHA epoxidations. It ensued that 10 lM of these two x3 fatty acids decreased by more than 80% and 60%, respectively, the formation by CYP2C9 of AA-epoxidised derivatives. These findings suggest that some physiological effects of x3 fatty acids may be due to a shift in the generation of active epoxidised metabolites of AA through CYP-mediated catalysis.
Cloning and Expression of Murine CYP2Cs and Their Ability to Metabolize Arachidonic Acid
Archives of Biochemistry and Biophysics, 1998
Five murine cytochrome P450 (CYP) 2C cDNAs were cloned and characterized, including four new members of this subfamily: CYP2C37, CYP2C38, CYP2C39, and CYP2C40. The cDNAs ranged from 1716 to 1812 bp in length and encoded polypeptides of 490 amino acid residues except for CYP2C40, which contained an additional glutamic acid residue at the carboxyl terminus. The amino acid identity of the murine CYP2Cs ranged from 69 to 92%, while the overall amino acid identity was 60%; however, within the six putative substrate recognition sites the identity was only 25 to 41%, suggesting possible differences in substrate specificity and product profiles. The CYP2C cDNAs were expressed in Escherichia coli following modification of the N-terminus. All five recombinant CYP2Cs metabolized arachidonic acid, but with different metabolic profiles and catalytic rates. Based on coelution with authentic standards on reverse-phase HPLC, the major metabolites were tentatively identified as follows: CYP2C29 and CYP2C39 produced 14,15-cisepoxyeicosatrienoic acid (EET); CYP2C37 produced 12-hydroxyeicosatetraenoic acid (HETE); CYP2C38 produced 11,12-EET; and CYP2C40 produced an unidentified metabolite that coeluted with 16-,17-, and 18-HETEs. The turnover numbers for CYP2C29, CYP2C37, CYP2C38, CYP2C39, and CYP2C40 were 0.34, 1.12, 5.15, 0.51, and 0.15 nmol/nmol/min, respectively. Reverse transcriptase-polymerase chain reaction demonstrated the presence of CYP2C29 mRNA in liver as well as in extrahepatic tissues including brain, kidney, lung, heart, and intestine. CYP2C38 and CYP2C40 were found in liver, brain, kidney, and intestine, with trace amounts in lung and heart, while CYP2C37 and CYP2C39 appeared to be liver specific.