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Cytochrome P450 Enzymes in Oxygenation of Prostaglandin Endoperoxides and Arachidonic Acid

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Cytochrome P450 Enzymes in Oxygenation of Prostaglandin Endoperoxides and Arachidonic Acid Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 231 _____________________________ _____________________________ Cytochrome P450 Enzymes in Oxygenation of Prostaglandin Endoperoxides and Arachidonic Acid Cloning, Expression and Catalytic Properties of CYP4F8 and CYP4F21 BY JOHAN BYLUND ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2000 Dissertation for the Degree of Doctor of Philosophy (Faculty of Pharmacy) in Pharmaceutical Pharmacology presented at Uppsala University in 2000 ABSTRACT Bylund, J. 2000. Cytochrome P450 Enzymes in Oxygenation of Prostaglandin Endoperoxides and Arachidonic Acid: Cloning, Expression and Catalytic Properties of CYP4F8 and CYP4F21. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from Faculty of Pharmacy 231 50 pp. Uppsala. ISBN 91-554-4784-8. Cytochrome P450 (P450 or CYP) is an enzyme system involved in the oxygenation of a wide range of endogenous compounds as well as foreign chemicals and drugs. This thesis describes investigations of P450-catalyzed oxygenation of prostaglandins, linoleic and arachidonic acids. The formation of bisallylic hydroxy metabolites of linoleic and arachidonic acids was studied with human recombinant P450s and with human liver microsomes. Several P450 enzymes catalyzed the formation of bisallylic hydroxy metabolites. Inhibition studies and stereochemical analysis of metabolites suggest that the enzyme CYP1A2 may contribute to the biosynthesis of bisallylic hydroxy fatty acid metabolites in adult human liver microsomes. 19R-Hydroxy-PGE and 20-hydroxy-PGE are major components of human and ovine semen, respectively. They are formed in the seminal vesicles, but the mechanism of their biosynthesis is unknown. Reverse transcription-polymerase chain reaction using degenerate primers for mammalian CYP4 family genes, revealed expression of two novel P450 genes in human and ovine seminal vesicles. The full coding regions of the genes were cloned and the enzymes were expressed in a yeast system. The human enzyme was designated CYP4F8 and the ovine enzyme was designated CYP4F21. Comparison of their deduced protein sequences showed that they had 74 % amino acid identity. Recombinant CYP4F8 oxygenated two prostaglandin endoperoxides (PGH1 and PGH2) and three stable PGH2 analogues into 19-hydroxy metabolites. Oxygenation of these substrates was mirrored when incubated with microsomes isolated from human seminal vesicles. These results suggest that CYP4F8 is present in human seminal vesicles and that 19R-hydroxy-PGE is formed by CYP4F8-catalyzed 19R-hydroxylation of PGH1 and PGH2, followed by PGE synthase-catalyzed isomerization. Studies of catalytic properties of recombinant CYP4F21 suggest that 20-hydroxy- PGE may be formed by similar mechanisms in ovine seminal vesicles. CYP4F8 is the first enzyme shown to hydroxylate prostaglandin endoperoxides. Johan Bylund, Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Biomedical Centre, Box-591, SE-751 24 Uppsala, Sweden © Johan Bylund 2000 ISSN 0282-7484 ISBN 91-554-4784-8 Printed in Sweden by Universitetstryckeriet, Ekonomikum, Uppsala, 2000 2 3 List of original papers This thesis is based on the following papers, which will be referred to by their Roman numerals in the text. I. Bylund, J., Kunz, T., Valmsen, K., and Oliw, E. H. (1998) Cytochrome P450 with Bisallylic Hydroxylation Activity on Arachidonic and Linoleic Acids Studied with Human Recombinant Enzymes and with Human and Rat Liver Microsomes. J. Pharmacol. Exp. Ther. 284, 51-60. II. Bylund, J., Ericsson, J. and Oliw, E. H. (1998) Analysis of Cytochrome P450 Metabolites of Arachidonic and Linoleic Acids by Liquid Chromatography-Mass Spectrometry with Ion Trap MS2. Anal. Biochem. 265, 55-68. III. Bylund, J., Finnström, N. and Oliw, E. H. (1999) Gene Expression of a Novel Cytochrome P450 of the CYP4F Subfamily in Human Seminal Vesicles. Biochem. Biophys. Res. Commun. 261, 169-174. IV. Bylund, J., Hidestrand, M., Ingelman-Sundberg, M. and Oliw, E. H. (2000) Identification of CYP4F8 in Human Seminal Vesicles as a Prominent 19-Hydroxylase of Prostaglandin Endo- peroxides. J. Biol. Chem. 275, 21844-21849. V. Bylund, J. and Oliw, E. H. (2000) Characterization of a Prostaglandin ω-Hydroxylase of Ram Seminal Vesicles: cDNA Cloning and Expression of CYP4F21. Manuscript The articles are reprinted with permission from the copyright holders. 4 TABLE OF CONTENTS INTRODUCTION ……………………………………………………………………. 7 Cytochrome P450 ...………………………………………………………………. 7 Reactions ………………………………………………………………………8 Expression and substrate specificity ...……………………………………….. 9 Eicosanoid biosynthesis ...……………………………………………………… 10 Prostaglandin H synthase pathway ..……………………………………….. 11 Lipoxygenase pathway ..……………………………………………………. 12 Cytochrome P450 pathway ..………………………………………………. 13 Hydroxylation of the ω-side chain ..…………………………………… 14 Epoxidation …………………………………………………………….15 Bisallylic hydroxylation ……………………………………………….. 16 Hydroxylation with double bond migration ..…………………………. 17 Eicosanoid metabolism ...………………………………………………………. 17 Seminal prostaglandins ..……………………………………………………. 18 AIMS …………………………………………………………………………………. 21 COMMENTS ON METHODOLOGY …………………………………………….. 22 Analysis of metabolites ...………………………………………………………... 22 Degenerate primers……………………………………………………………….. 22 Recombinant cytochrome P450 ...………………………………………………... 23 PGH2 and stable PGH2 analogues …………………………………………….…. 24 RESULTS ……………………………………………………………………………. 25 Bisallylic hydroxylation of fatty acids…...………………………………………. 25 Identification of fatty acid metabolites with LC-MS …………………..………... 26 Gene expression of P450s in human seminal vesicles …………………………….27 Catalytic properties of CYP4F8 ……………………………………………….....27 CYP4F21 in ovine seminal vesicles ……………………………………………… 29 DISCUSSION ………………………………………………………………………. 30 Bisallylic hydroxylation of fatty acids (papers I-II) …………………………..… 30 Biosynthesis and metabolism of seminal prostaglandins (papers III-V) ……….... 32 CONCLUSIONS…………………………………………………………………..… 38 ACKNOWLEDGEMENTS…………………………………………..……………… 39 REFERENCES ………………………………………………………………………..40 5 ABBREVIATIONS APCI atmospheric pressure chemical ionization CYP cytochrome P450 EETepoxyeicosatrienoic acid ER endoplasmic reticulum ESI electrospray ionization DHET dihydroxyeicosatrienoic acid GC-MS gas chromatography-mass spectrometry HETE hydroxyeicosatetraenoic acid HODE hydroxyoctadecadienoic acid HPETE hydroperoxyeicosatetraenoic acid HPLC high performance liquid chromatography LC-MS liquid chromatography-mass spectrometry LTleukotriene P450 cytochrome P450 PG prostaglandin PGH prostaglandin H PGG prostaglandin G PGI2 prostacyclin RP-HPLC reverse phase-high performance liquid chromatography RT-PCR reverse transcriptase-polymerase chain reaction SIM selective ion monitoring TXA2 thromboxane A2 12-HHT 12-hydroxyheptadecatrienoic acid 6 INTRODUCTION Arachidonic acid is a polyunsaturated fatty acid, which is present in most human and animal cells. Arachidonic acid can be converted into oxygenated metabolites, the so-called eicosanoids. The eicosanoids are involved in many physiological and pathophysiological functions such as blood pressure regulation, blood platelet aggregation, inflammation, reproduction and cancer. There are three major pathways involved in the formation of eicosanoids, the prostaglandin H synthase, the lipoxygenase, and the cytochrome P450 pathways. The understanding of the physiological functions of the eicosanoids and their mechanisms of formation has generated many new drugs and treatments of common disorders e.g. inflammation, pain, asthma and cardiovascular diseases. This study focuses on the involvement of cytochrome P450 enzymes in the biosynthesis of two groups of eicosanoids, hydroxyeicosatetraenoic acids and ω/(ω-1)-hydroxyprostaglandins. An increased knowledge of the mechanism of biosynthesis and metabolism of these eicosanoids may contribute to a better understanding of their physiological functions. Cytochrome P450 Cytochrome P450 (P450) was discovered in the late 1950’s (1, 2). P450 refers to a superfamily of heme-thiolate enzymes whose Fe2+-carbon monoxide complex shows an absorption spectrum with a maximum at 450 nm. The diversity of P450s has been found to be enormous. The P450 enzymes can be found in virtually all organisms including bacteria, fungi, yeast, plants, insects, fish and mammals. All P450 genes have probably evolved from one single ancestral gene, which existed before the time of prokaryote/eukaryote divergence (3). Due to the large number of P450s, a standardized nomenclature system has been developed. The enzymes have been organized on the basis of identities in protein sequence. The enzymes are named CYP, representing cytochrome P450, followed by a number denoting the family, a letter designating the subfamily and a second numeral representing the individual enzyme. The corresponding gene name is written in italics. Enzymes that have >40 % amino acid sequence identity are considered to belong to the same family, whereas enzymes with >55 % sequence identity are in the same subfamily (3). The P450 enzymes have two major functions (3, 4). They are involved in the biosynthesis, bio-activation and catabolism of endogenous compounds, such as fatty acids, steroid hormones, vitamins, bile acids, and eicosanoids. They are also involved in the metabolism of foreign chemicals and drugs. Drugs and other chemicals are often converted by P450 to more polar compounds that can be directly excreted or further conjugated by other enzymes. The conjugate makes the
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