Alternative splicing determines the function of CYP4F3 by switching substrate specificity.

Diversity of cytochrome P450 function is determined by the expression of multiple genes, many of which have a high degree of identity. We report that the use of alternate exons, each coding for 48 amino acids, generates isoforms of human CYP4F3 that differ in substrate specificity, tissue distribution, and biological function. Both isoforms contain a total of 520 amino acids. CYP4F3A, which incorporates exon 4, inactivates LTB4 by omega-hydroxylation (Km = 0.68 microm) but has low activity for arachidonic acid (Km = 185 microm); it is the only CYP4F isoform expressed in myeloid cells in peripheral blood and bone marrow. CYP4F3B incorporates exon 3 and is selectively expressed in liver and kidney; it is also the predominant CYP4F isoform in trachea and tissues of the gastrointestinal tract. CYP4F3B has a 30-fold higher Km for LTB4 compared with CYP4F3A, but it utilizes arachidonic acid as a substrate for omega-hydroxylation (Km = 22 microm) and generates 20-HETE, an activator of protein kinase C and Ca2+/calmodulin-dependent kinase II. Homology modeling demonstrates that the alternative exon has a position in the molecule which could enable it to contribute to substrate interactions. The results establish that tissue-specific alternative splicing of pre-mRNA can be used as a mechanism for changing substrate specificity and increasing the functional diversity of cytochrome P450 genes.     

Human CYP4F3s are the main catalysts in the oxidation of fatty acid epoxides

CYP4F isoforms are involved in the oxidation of important cellular mediators such as leukotriene B4 (LTB4) and prostaglandins. The proinflammatory agent LTB4 and cytotoxic leukotoxins have been associated with several inflammatory diseases. We present evidence that the hydroxylation of Z 9(10)-epoxyoctadecanoic, Z 9(10)-epoxyoctadec-Z 12-enoic, and Z 12(13)-epoxyoctadec-Z 9-enoic acids and that of monoepoxides from arachidonic acid [epoxyeicosatrienoic acid (EET)] is important in the regulation of leukotoxin and EET activity. These three epoxidized derivatives from the C18 family (C18-epoxides) were converted to 18-hydroxy-C18-epoxides by human hepatic microsomes with apparent Km values of between 27.6 and 175 microM. Among recombinant P450 enzymes, CYP4F2 and CYP4F3B catalyzed mainly the omega-hydroxylation of C18-epoxides with an apparent Vmax of between 0.84 and 15.0 min(-1), whereas the apparent Vmax displayed by CYP4F3A, the isoform found in leukocytes, ranged from 3.0 to 21.2 min(-1). The rate of omega-hydroxylation by CYP4A11 was experimentally found to be between 0.3 and 2.7 min(-1). CYP4F2 and CYP4F3 exhibited preferences for omega-hydroxylation of Z 8(9)-EET, whereas human liver microsomes preferred Z 11(12)-EET and, to a lesser extent, Z 8(9)-EET. Moreover, vicinal diol from both C18-epoxides and EETs were omega-hydroxylated by liver microsomes and by CYP4F2 and CYP4F3. These data support the hypothesis that the human CYP4F subfamily is involved in the omega-hydroxylation of fatty acid epoxides. These findings demonstrate that another pathway besides conversion to vicinal diol or chain shortening by beta-oxidation exists for fatty acid epoxide inactivation.     

Cytochrome P450 omega-hydroxylase pathway of tocopherol catabolism. Novel mechanism of regulation of vitamin E status

Postabsorptive elimination of the various forms of vitamin E appears to play a key role in regulation of tissue tocopherol concentrations, but mechanisms of tocopherol metabolism have not been elucidated. Here we describe a pathway involving cytochrome P450-mediated omega-hydroxylation of the tocopherol phytyl side chain followed by stepwise removal of two- or three-carbon moieties, ultimately yielding the 3'-carboxychromanol metabolite that is excreted in urine. All key intermediates of gamma-tocopherol metabolism via this pathway were identified in hepatocyte cultures using gas chromatography-mass spectrometry. NADPH-dependent synthesis of the initial gamma- and alpha-tocopherol 13'-hydroxy and -carboxy metabolites was demonstrated in rat and human liver microsomes. Functional analysis of several recombinant human liver P450 enzymes revealed that tocopherol-omega-hydroxylase activity was associated only with CYP4F2, which also catalyzes omega-hydroxylation of leukotriene B(4) and arachidonic acid. Tocopherol-omega-hydroxylase exhibited similar binding affinities but markedly higher catalytic activities for gamma-tocopherol than alpha-tocopherol, suggesting a role for this pathway in the preferential physiological retention of alpha-tocopherol and elimination of gamma-tocopherol. Sesamin potently inhibited tocopherol-omega-hydroxylase activity exhibited by CYP4F2 and rat or human liver microsomes. Since dietary sesamin also results in elevated tocopherol levels in vivo, this pathway appears to represent a functionally significant means of regulating vitamin E status.     

Influence of major structural features of tocopherols and tocotrienols on their omega-oxidation by tocopherol-omega-hydroxylase

Human cytochrome P450 4F2 (CYP4F2) catalyzes the initial omega-hydroxylation reaction in the metabolism of tocopherols and tocotrienols to carboxychromanols and is, to date, the only enzyme shown to metabolize vitamin E. The objective of this study was to characterize this activity, particularly the influence of key features of tocochromanol substrate structure. The influence of the number and positions of methyl groups on the chromanol ring, and of stereochemistry and saturation of the side chain, were explored using HepG2 cultures and microsomal reaction systems. Human liver microsomes and microsomes selectively expressing recombinant human CYP4F2 exhibited substrate activity patterns similar to those of HepG2 cells. Although activity was strongly associated with substrate accumulation by cells or microsomes, substantial differences in specific activities between substrates remained under conditions of similar microsomal membrane substrate concentration. Methylation at C5 of the chromanol ring was associated with markedly low activity. Tocotrienols exhibited much higher Vmax values than their tocopherol counterparts. Side chain stereochemistry had no effect on omega-hydroxylation of alpha-tocopherol (alpha-TOH) by any system. Kinetic analysis of microsomal CYP4F2 activity revealed Michaelis-Menten kinetics for alpha-TOH but allosteric cooperativity for other vitamers, especially tocotrienols. Additionally, alpha-TOH was a positive effector of omega-hydroxylation of other vitamers. These results indicate that CYP4F2-mediated tocopherol-omega-hydroxylation is a central feature underlying the different biological half-lives, and therefore biopotencies, of the tocopherols and tocotrienols.     

Omega-oxidation of very long-chain fatty acids in human liver microsomes. Implications for X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a severe neurodegenerative disorder biochemically characterized by elevated levels of very long-chain fatty acids (VLCFA). Excess levels of VLCFAs are thought to play an important role in the pathogenesis of X-ALD. Therefore, therapeutic approaches for X-ALD are focused on the reduction or normalization of VLCFAs. In this study, we investigated an alternative oxidation route for VLCFAs, namely omega-oxidation. The results described in this study show that VLCFAs are substrates for the omega-oxidation system in human liver microsomes. Moreover, VLCFAs were not only converted into omega-hydroxy fatty acids, but they were also further oxidized to dicarboxylic acids via cytochrome P450-mediated reactions. High sensitivity toward the specific P450 inhibitor 17-octadecynoic acid suggested that omega-hydroxylation of VLCFAs is catalyzed by P450 enzymes belonging to the CYP4A/F subfamilies. Studies with individually expressed human recombinant P450 enzymes revealed that two P450 enzymes, i.e. CYP4F2 and CYP4F3B, participate in the omega-hydroxylation of VLCFAs. Both enzymes belong to the cytochrome P450 4F subfamily and have a high affinity for VLCFAs. In summary, this study demonstrates that VLCFAs are substrates for the human omega-oxidation system, and for this reason, stimulation of the in vivo VLCFA omega-oxidation pathway may provide an alternative mode of treatment to reduce the levels of VLCFAs in patients with X-ALD.     

Evaluation of cytochrome P450 4 family as mediator of phospholipase D activation in aortic vascular smooth muscle cells

Norepinephrine (NE) stimulates phospholipase D (PLD) activity via phospholipase A2-dependent arachidonic acid release in rabbit aortic vascular smooth muscle cells (VSMC). We have previously shown that exogenous 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid generated through the cytochrome P450 (CYP) 4A pathway in vivo, stimulates PLD activity. Whether endogenous CYP4-derived arachidonic acid metabolites act as intracellular mediators of NE-induced PLD activation in VSMC is not known. In rabbit aortic VSMC, prototypical hepatic/renal CYP4A inducers such as fenofibrate and Wy 14643 inhibited both basal and NE-induced PLD activity after 48 h of exposure. The level of CYP4F, and to a lesser extent CYP4A, was also decreased by these agents. The expression levels of rabbit aortic VSMC CYP4A and CYP4F isoforms were reduced by antisense oligonucleotides treatment for 48 hours as measured by RTQ-PCR or Western blotting. This reduction in CYP4A or CYP4F levels did not change NE-induced PLD activation. The corresponding CYP4A scrambled and CYP4F sense oligonucleotides did not alter CYP levels. PLD activity was increased by ~70% after 15 min of stimulation with NE, whereas lauric acid omega-hydroxylase activity, a measure of fatty acid omega-hydroxylation, was unchanged. Inhibition of omega-hydroxylation with DDMS and HET0016, selective omega-hydroxylase inhibitors, and 20-HEDE, an antagonist of 20-HETE, increased PLD activity in a concentration-dependent manner and did not alter NE-induced PLD activation. These data suggest that PLD activation by NE is independent of the CYP4A/4F enzymes in rabbit aortic VSMC.     

Purification and characterization of recombinant rat hepatic CYP4F1.

CYP4F1 was discovered by Chen and Hardwick (Arch. Biochem. Biophys. 300, 18-23, 1993) as a new CYP4 cytochrome P450 (P450) preferentially expressed in rat hepatomas. However, the catalytic function of this P450 remained poorly defined. We have purified recombinant CYP4F1 protein to a specific content of 12 nmol of P450/mg of protein from transfected yeast cells by chromatography of solubilized microsomes on an amino-n-hexyl Sepharose 4B column, followed by sequential HPLC on a DEAE column and two hydroxylapatite columns. The purified P450 was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 53 kDa. The enzyme catalyzed the omega-hydroxylation of leukotriene B(4) with a K(m) of 134 microM and a V(max) of 6.5 nmol/min/nmol of P450 in the presence of rabbit hepatic NADPH-P450 reductase and cytochrome b(5). In addition, 6-trans-LTB(4), lipoxin A(4), prostaglandin A(1), and several hydroxyeicosatetraenoic acids (HETEs) were also omega-hydroxylated. Of several eicosanoids examined, 8-HETE was the most efficient substrate, with a K(m) of 18.6 microM and a V(max) of 15.8 nmol/min/nmol of P450. In contrast, no activity was detected toward lipoxin B(4), laurate, palmitate, arachidonate, and benzphetamine. The results suggest that CYP4F1 participates in the hepatic inactivation of several bioactive eicosanoids.     

Identification of the cytochrome P450 enzymes responsible for the omega-hydroxylation of phytanic acid

Patients suffering from Refsum disease have a defect in the alpha-oxidation pathway which results in the accumulation of phytanic acid in plasma and tissues. Our previous studies have shown that phytanic acid is also a substrate for the omega-oxidation pathway. With the use of specific inhibitors we now show that members of the cytochrome P450 (CYP450) family 4 class are responsible for phytanic acid omega-hydroxylation. Incubations with microsomes containing human recombinant CYP450s (Supersomes) revealed that multiple CYP450 enzymes of the family 4 class are able to omega-hydroxylate phytanic acid with the following order of efficiency: CYP4F3A>CYP4F3B>CYP4F2>CYP4A11.     

Formation of 20-hydroxyeicosatetraenoic acid, a vasoactive and natriuretic eicosanoid, in human kidney. Role of Cyp4F2 and Cyp4A11

20-hydroxyeicosatetraenoic acid (20-HETE), an omega-hydroxylated arachidonic acid (AA) metabolite, elicits specific effects on kidney vascular and tubular function that, in turn, influence blood pressure control. The human kidney's capacity to convert AA to 20-HETE is unclear, however, as is the underlying P450 catalyst. Microsomes from human kidney cortex were found to convert AA to a single major product, namely 20-HETE, but failed to catalyze AA epoxygenation and midchain hydroxylation. Despite the monophasic nature of renal AA omega-hydroxylation kinetics, immunochemical studies revealed participation of two P450s, CYP4F2 and CYP4A11, since antibodies to these enzymes inhibited 20-HETE formation by 65. 9 +/- 17 and 32.5 +/- 14%, respectively. Western blotting confirmed abundant expression of these CYP4 proteins in human kidney and revealed that other AA-oxidizing P450s, including CYP2C8, CYP2C9, and CYP2E1, were not expressed. Immunocytochemistry showed CYP4F2 and CYP4A11 expression in only the S2 and S3 segments of proximal tubules in cortex and outer medulla. Our results demonstrate that CYP4F2 and CYP4A11 underlie conversion of AA to 20-HETE, a natriuretic and vasoactive eicosanoid, in human kidney. Considering their proximal tubular localization, these P450 enzymes may partake in pivotal renal functions, including the regulation of salt and water balance, and arterial blood pressure itself.     

A novel human cytochrome P450 4F isoform (CYP4F11): cDNA cloning, expression, and genomic structural characterization

By a combination of cDNA library screening and rapid amplification of cDNA ends analysis, a novel human cytochrome P450 4F isoform has been cloned and sequenced. The new 4F isoform is designated CYP4F11 and contains 1765 nucleotides. The coding region encodes 524 amino acid residues, and the heme-binding region is highly conserved. The CYP4F11 amino acid sequence has 80.0, 82.3, and 79.2% identity to CYP4F2, CYP4F3, and CYP4F8 amino acid sequences, respectively. In vitro translation shows the molecular mass of CYP4F11 is approximately 57 kDa, consistent with the calculated molecular mass. CYP4F11 is expressed mainly in human liver, followed by kidney, heart, and skeletal muscle. The genomic structure of CYP4F11 was solved by database searching and computer analysis. The coding region of CYP4F11 has 12 exons. The CYP4F11 gene is located 16 kb upstream of the CYP4F2 gene on chromosome 19. This is consistent with the notion that the human cytochrome P450 4F genes form a cluster on chromosome 19.     

Cloning and expression of three rabbit kidney cDNAs encoding lauric acid omega-hydroxylases

cDNAs encoding three cytochrome P-450 enzymes were cloned from a rabbit kidney cDNA library. These three cDNAs exhibit greater than 90% nucleotide sequence identity across the coding region. This degree of sequence identity is also seen with P450IVA4, an enzyme that catalyzes the omega-hydroxylation of prostaglandins and that is elevated during pregnancy and induced by progesterone in rabbit lung. The 3' untranslated regions of the three cDNAs display very little sequence identity, suggesting that they are the products of distinct genes. The predicted amino acid sequences derived from each cDNA and for P450IVA4 exhibit about 85% identity. Each cDNA was inserted into an expression vector for transient transfection of COS-1 cells. The transfected cells each expressed a protein recognized by antibodies to P450IVA4. Microsomes isolated from the cells transfected with each cDNA efficiently catalyzed the omega-hydroxylation of lauric acid with rates that greatly exceed that catalyzed by microsomes isolated from the host cell line. One of the cDNAs encodes an enzyme that omega-hydroxylates prostaglandin A1; however, the specific activity was 2 orders of magnitude lower than that for lauric acid. Our results indicate that the substrate selectivity of the kidney P-450s encoded by these cDNAs is distinct from that of the lung P450IVA4 and that multiple enzymes comprise P-450 class IVA in the rabbit.     

Distinction between human cytochrome P450 (CYP) isoforms and identification of new phosphorylation sites by mass spectrometry

In mammals, Cytochrome P450 (CYP) enzymes are bound to membranes of the endoplasmic reticulum and mitochondria, where they are responsible for the oxidative metabolism of many xenobiotics as well as organic endogenous compounds. In humans, 57 isoforms were identified which are classified based on sequence homology. In the present work, we demonstrate the performance of a mass spectrometry-based strategy to simultaneously detect and differentiate distinct human Cytochrome P450 (CYP) isoforms including the highly similar CYP3A4, CYP3A5, CYP3A7, as well as CYP2C8, CYP2C9, CYP2C18, CYP2C19, and CYP4F2, CYP4F3, CYP4F11, CYP4F12. Compared to commonly used immunodetection methods, mass spectrometry overcomes limitations such as low antibody specificity and offers high multiplexing possibilities. Furthermore, CYP phosphorylation, which may affect various biochemical and enzymatic properties of these enzymes, is still poorly analyzed, especially in human tissues. Using titanium dioxide resin combined with tandem mass spectrometry for phosphopeptide enrichment and sequencing, we discovered eight human P450 phosphorylation sites, seven of which were novel. The data from surgical human liver samples establish that the isoforms CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C8, CYP2D6, CYP3A4, CYP3A7, and CYP8B1 are phosphorylated in vivo. These results will aid in further investigation of the functional significance of protein phosphorylation for this important group of enzymes.     

Characterization of human liver leukotriene B(4) omega-hydroxylase P450 (CYP4F2)

We previously reported the cloning of a human liver leukotriene B(4) (LTB(4)) omega-hydroxylase P450 designated CYP 4F2 [Kikuta et al. (1994) FEBS Lett. 348, 70-74]. However, the properties of CYP 4F2 remain poorly defined. The preparation solubilized using n-octyl-beta-D-glucopyranoside from microsomes of CYP 4F2-expressing yeast cells catalyzes v- hydroxylation of LTB(4), 6-trans-LTB(4), lipoxin A(4), 8-hydroxyeicosatetraenoate, 12-hydroxyeicosatetraenoate, and 12-hydroxystearate in the presence of rabbit liver NADPH-P450 reductase. In addition, the enzyme shows ethoxycoumarin O-deethylase and p-nitroanisole O-demethylase activities. The enzyme was purified to apparent electrophoretic homogeneity from yeast cells by sequential chromatography of solubilized microsomes through amino-n-hexyl-Sepharose 4B, DEAE-HPLC, and hydroxylapatite HPLC columns. The final preparation showed a specific content of 11.1 nmol of P450/mg of protein, with an apparent molecular mass of 56.3 kDa. CYP 4F2 was distinguished from the closely homologous CYP 4F3 (human neutrophil LTB(4) omega-hydroxylase) by its much higher K(m) for LTB(4), inability to omega-hydroxylate lipoxin B(4), and extreme instability.     

Expression of CYP4F2 in human liver and kidney: assessment using targeted peptide antibodies

P450 enzymes comprising the human CYP4F gene subfamily are catalysts of eicosanoid (e.g., 20-HETE and leukotriene B4) formation and degradation, although the role that individual CYP4F proteins play in these metabolic processes is not well defined. Thus, we developed antibodies to assess the tissue-specific expression and function of CYP4F2, one of four CYP4F P450s found in human liver and kidney. Peptide antibodies elicited in rabbits to CYP4F2 amino acid residues 61-74 (WGHQGMVNPTEEG) and 65-77 (GMVNPTEEGMRVL) recognized on immunoblots only CYP4F2 and not CYP4F3b, CYP4F11 or CYP4F12. Immunoquantitation with anti-CYP4F2 peptide IgG showed highly variable CYP4F2 expression in liver (16.4+/-18.6pmol/mg microsomal protein; n=29) and kidney cortex (3.9+/-3.8 pmol/mg; n=10), with two subjects lacking the hepatic or renal enzyme entirely. CYP4F2 content in liver microsomes was significantly correlated (r> or =0.63; p<0.05) with leukotriene B4 and arachidonate omega-hydroxylase activities, which are both CYP4F2-catalyzed. Our study provides the first example of a peptide antibody that recognizes a single CYP4F P450 expressed in human liver and kidney, namely CYP4F2. Immunoquantitation and correlation analyses performed with this antibody suggest that CYP4F2 functions as a predominant LTB4 and arachidonate omega-hydroxylase in human liver.     

Expression of human aromatase (CYP19) in Escherichia coli by N-terminal replacement and induction of cold stress response

CYP19 (P450arom) catalyzes the aromatization reaction of C19 steroids leading to estrogens. While readily expressed in insect cells, the human P450arom has been a difficult P450 to express in Escherichia coli at useful levels. In the present study, we replaced the N-terminal sequence in human CYP19 with the corresponding sequences of other microsomal P450s (CYP2C11 and CYP17) that are efficiently expressed in E. coli. Although the N-terminal replacement alone was not sufficient for the expression, human P450arom was successfully expressed up to the level of 240nmol/l culture by the combination of the N-terminal replacement and the induction of cold stress response by 1 microg/ml chloramphenicol. Membrane fractions containing the expressed P450arom catalyzed aromatization of androstenedione with a specific activity of 4.9 nmol/min/nmol P450. Our results are important to provide large quantities of human P450arom as an active form for structure-function studies.     

NADPH- and hydroperoxide-supported 17beta-estradiol hydroxylation catalyzed by a variant form (432L, 453S) of human cytochrome P450 1B1

Human cytochrome P450 1B1 (CYP1B1) catalyzes the hydroxylation of 17beta-estradiol (E(2)) at C-4, with a lesser activity at C-2. The E(2) 4-hydroxylase activity of human CYP1B1 was first observed in studies of MCF-7 breast cancer cells. Sequencing of polymerase chain reaction products revealed that CYP1B1 expressed in MCF-7 cells was not the previously characterized enzyme but a polymorphic form with leucine substituted for valine at position 432 and serine substituted for asparagine at position 453. To investigate the NADPH- and organic hydroperoxide-supported E(2) hydroxylase activities of the 432L, 453S form of human CYP1B1, the MCF-7 CYP1B1 cDNA was cloned and the enzyme was expressed in Sf9 insect cells. In microsomal assays supplemented with human NADPH:cytochrome P450 oxidoreductase, the expressed 432L, 453S form catalyzed NADPH-supported E(2) hydroxylation with a similar preference for 4-hydroxylation as the 432V, 453N form, with maximal rates of 1.97 and 0.37 nmol (min)(-1)(nmol cytochrome P450)(-1) for 4- and 2-hydroxylation, respectively. Cumeme hydroperoxide efficiently supported E(2) hydroxylation by both the 432V, 453N and 432L, 453S forms at several-fold higher rates than the NADPH-supported activities and with a lesser preference for E(2) 4- versus 2-hydroxylation (2:1). The hydroperoxide-supported activities of both forms were potently inhibited by the CYP1B1 inhibitor, 3,3',4, 4',5,5'-hexachlorobiphenyl. These results indicate that the 432V, 453N and 432L, 453S forms of CYP1B1 have similar catalytic properties for E(2) hydroxylation, and that human CYP1B1 is very efficient in catalyzing the hydroperoxide-dependent formation of catecholestrogens.     

Omega oxidation of 3-hydroxy fatty acids by the human CYP4F gene subfamily enzyme CYP4F11

Long-chain 3-hydroxydicarboxylic acids (3-OHDCAs) are thought to arise via beta-oxidation of the corresponding dicarboxylic acids (DCAs), although long-chain DCAs are neither readily transported into nor beta-oxidized in mitochondria. We thus examined whether omega-hydroxylation of 3-hydroxy fatty acids (3-OHFAs), formed via incomplete mitochondrial oxidation, is a more likely pathway for 3-OHDCA production. NADPH-fortified human liver microsomes converted 3-hydroxystearate and 3-hydroxypalmitate to their omega-hydroxylated metabolites, 3,18-dihydroxystearate and 3,16-dihydroxypalmitate, respectively, as identified by GC-MS. Rates of 3,18-dihydroxystearate and 3,16-dihydroxypalmitate formation were 1.23 +/- 0.5 and 1.46 +/- 0.30 nmol product formed/min/mg protein, respectively (mean +/- SD; n = 13). Polyspecific CYP4F antibodies markedly inhibited microsomal omega-hydroxylation of 3-hydroxystearate (68%) and 3-hydroxypalmitate (99%), whereas CYP4A11 and CYP2E1 antibodies had little effect. Upon reconstitution, CYP4F11 and, to a lesser extent, CYP4F2 catalyzed omega-hydroxylation of 3-hydroxystearate, whereas CYP4F3b, CYP4F12, and CYP4A11 exhibited negligible activity. CYP4F11 was the lone CYP4F/A enzyme that effectively oxidized 3-hydroxypalmitate. Kinetic parameters of microsomal 3-hydroxystearate metabolism were K(m) = 55 microM and V(max) = 8.33 min(-1), whereas those for 3-hydroxypalmitate were K(m) = 56.4 microM and V(max) = 14.2 min(-1). CYP4F11 kinetic values resembled those of native microsomes, with K(m) = 53.5 microM and V(max) = 13.9 min(-1) for 3-hydroxystearate and K(m) = 105.8 microM and V(max) = 70.6 min(-1) for 3-hydroxypalmitate. Our data show that 3-hydroxystearate and 3-hydroxypalmitate are converted to omega-hydroxylated 3-OHDCA precursors in human liver and that CYP4F11 is the predominant catalyst of this reaction. CYP4F11-promoted omega-hydroxylation of 3-OHFAs may modulate the disposition of these compounds in pathological states in which enhanced fatty acid mobilization or impairment of mitochondrial fatty acid beta-oxidation increases circulating 3-OHFA levels.