cDNA Cloning and Expression of CYP4F12, a Novel Human Cytochrome P450 Johan Bylund, 1 Mattias Bylund, and Ernst H. Oliw Department of Pharmaceutical Biosciences, BMC, P.O. Box 591, SE-751 24 Uppsala, Sweden Received December 20, 2000 cDNA of a novel human cytochrome P450 was cloned from human liver by reverse transcription-polymerase chain reaction and designated CYP4F12. The open read- ing frame coded for 524 amino acids, and the sequence could be aligned with 78 – 83% amino acid identity to the four human CYP4F enzymes (CYP4F2, CYP4F3, CYP4F8 and CYP4F11). Northern blot analysis suggested three major transcripts of CYP4F12, which were detected in liver, kidney, colon, small intestine and heart. The CYP4F12 gene contained 13 exons and was located at chromosome 19p13.1. CYP4F12, expressed in yeast, oxi- dized arachidonic acid to 18-hydroxyarachidonic acid, and the -side chain of two stable prostaglandin (PG) H 2 analogs (11,9-epoxymethano-PGH 2 and 9,11-diazo-15- deoxy-PGH 2 ). CYP4F12 oxidized the -side chain of leukotriene B 4 , PGE 2 , PGF 2 , PGH 2 , and 9,11-epoxy- methano-PGH 2 poorly. Several CYP4F enzymes are im- portant 1- and 2-hydroxylases of eicosanoids. The physiological function of CYP4F12 merits further investigation. © 2001 Academic Press Cytochrome P450 enzymes are present in many tis- sues and can oxidize xenobiotics as well as endogenous compounds of physiological importance, e.g., vitamin D, cholesterol, bile acids and eicosanoids (1). The cyto- chrome P450 enzymes are divided into families and subfamilies based on amino acid similarities. The CYP4 family contains important -side chain hydroxy- lases of fatty acids and eicosanoids (1). Within this family, the CYP4F subfamily is of particular interest for biosynthesis and metabolism of structurally differ- ent eicosanoids (2– 8). Four human CYP4F members have been identified to date, CYP4F2, CYP4F3, CYP4F8 and CYP4F11 (4, 9 –11). CYP4F3 is expressed in polymorphonuclear leukocytes and catalyzes 20-hydroxylation of a po- tent chemotactic mediator, LTB 4 , with a K m below 1 M (4). CYP4F8 catalyzes 19-hydroxylation of PGH 1 and PGH 2 in the seminal vesicles, a key step in the biosynthesis of seminal 19-hydroxy-PGE (5). 19- Hydroxy-PGE 1 and 19-hydroxy-PGE 2 of human se- men are proposed to be important immunomodula- tors in the female genital tract (12). Less is known about the physiological function of CYP4F2 and CYP4F11. CYP4F2 is expressed in liver and kidney (3, 9, 13). Recombinant CYP4F2 catalyzes 20- hydroxylation of LTB 4 with a high K m of about 60 M (2). Purified hepatic CYP4F2, or a highly related enzyme, has also been implicated in 20-hydroxyl- ation of arachidonic acid in the liver and kidney (3, 13). Recently a new CYP4F gene, CYP4F11, was identified (11). CYP4F11 is expressed in the kidney, liver and some other tissues, but its catalytic prop- erty is unknown. CYP4F2, CYP4F3, CYP4F8 and CYP4F11 are all located in a gene cluster on chromosome 19p13.1 (10, 11, 14, 15). These four CYP4F genes are structurally very similar and are apparently formed by gene dupli- cation. We searched the GenBank with cDNA of CYP4F8 and found that at least one additional CYP4F gene may be present in the CYP4F gene cluster on chromosome 19. Similar observations have been re- ported by Nelson (16). The aims of the present study were to clone the cDNA of the uncharacterized CYP4F gene, to deter- mine its genomic structure and gene expression in different human tissues. The gene was designated CYP4F12. Our final goal was to express CYP4F12 in yeast and to analyze its catalytic properties with ara- chidonic acid and some physiologically important eico- sanoids as substrates. The sequence reported in this paper has been deposited in Gen- Bank, Accession number AY008841. Abbreviations used: CYP, cytochrome P450; EST, expressed se- quence tag; HETE, hydroxyeicosatetraenoic acid; LC-MS, liquid chromatography-mass spectrometry; LTB 4 , leukotriene B 4 , MS/MS, tandem mass spectrometry; PG, prostaglandin; RT-PCR, reverse transcription-polymerase chain reaction; UTR, untranslated region. 1 To whom correspondence should be addressed. Fax: +46 18 55 2936. E-mail: Johan.Bylund@farmbio.uu.se. Biochemical and Biophysical Research Communications 280, 892– 897 (2001) doi:10.1006/bbrc.2000.4191, available online at http://www.idealibrary.com on 892 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.