Characterization of a cDNA encoding a human kidney, cytochrome 4A fatty acid ω-hydroxylase and the cognate enzyme expressed in Escherichia coli

Palmer, Colin N.A.; Richardson, Toby H.; Griffin, Keith J.; Hsu, Ming Lun; Muerhoff, A. Scott; Clark, J. E.; Johnson, Eric F.

doi:10.1016/0167-4781(93)90285-l

Cited by 71 publications

(24 citation statements)

References 30 publications

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“…Consistent with this notion, a homology model for 4A11 reveals a larger substrate-binding cavity when compared with 4B1 that accommodates longer fatty acids with the side chain of Arg-96 positioned on the second strand of the ␤-sheet 1 to facilitate the binding of lauric acid (Fig. 7) and longer fatty acids, such as palmitic and arachidonic acid, which are substrates for human CYP4A11 (51). Arg-96 of 4A11 is conserved at this alignment position in rabbit 4A4 and 4A5, rat 4A2, 4A3, and 4A8, and mouse 4a12a, 4a12b, and 4a14.…”

Section: Resultsmentioning

confidence: 74%

The Crystal Structure of Cytochrome P450 4B1 (CYP4B1) Monooxygenase Complexed with Octane Discloses Several Structural Adaptations for ω-Hydroxylation

Hsu

Baer

Rettie

et al. 2017

Journal of Biological Chemistry

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Edited by Ruma BanerjeeP450 family 4 fatty acid -hydroxylases preferentially oxygenate primary C-H bonds over adjacent, energetically favored secondary C-H bonds, but the mechanism explaining this intriguing preference is unclear. To this end, the structure of rabbit P450 4B1 complexed with its substrate octane was determined by X-ray crystallography to define features of the active site that contribute to a preference for -hydroxylation. The structure indicated that octane is bound in a narrow active-site cavity that limits access of the secondary C-H bond to the reactive intermediate. A highly conserved sequence motif on helix I contributes to positioning the terminal carbon of octane for -hydroxylation. Glu-310 of this motif auto-catalytically forms an ester bond with the heme 5-methyl, and the immobilized Glu-310 contributes to substrate positioning. The preference for -hydroxylation was decreased in an E310A mutant having a shorter side chain, but the overall rates of metabolism were retained. E310D and E310Q substitutions having longer side chains exhibit lower overall rates, likely due to higher conformational entropy for these residues, but they retained high preferences for octane -hydroxylation. Sequence comparisons indicated that active-site residues constraining octane for -hydroxylation are conserved in family 4 P450s. Moreover, the heme 7-propionate is positioned in the active site and provides additional restraints on substrate binding. In conclusion, P450 4B1 exhibits structural adaptations for -hydroxylation that include changes in the conformation of the heme and changes in a highly conserved helix I motif that is associated with selective oxygenation of unactivated primary C-H bonds.Cytochrome P450 family 4 fatty acid -hydroxylases are heme-containing monooxygenases that provide pathways for the reduction of potentially toxic non-esterified fatty acids and for the elimination of excess nutritive and non-nutritive lipids by catalyzing the addition of oxygen to a C-H bond of the terminal -carbon of fatty acids (1, 2). The resulting -alcohols are generally oxidized further to produce dicarboxylic acids, but they also may be transformed to glucuronides or esterified to glycerolipids. Urinary dicarboxylic fatty acids are elevated in humans by fasting or in uncontrolled diabetes, which are conditions where adipocyte lipolysis increases the availability of non-esterified fatty acids to fuel metabolism (3). It is estimated that roughly 15% of fatty acids undergo -hydroxylation during peak periods of fatty acid catabolism (4), whereas this fraction is much smaller under conditions where concentrations of nonesterified fatty acids are low (5). Collectively, these enzymes provide pathways for the metabolic clearance of branched chain fatty acids, eicosanoids, and xenobiotic substrates such as dietary phytanic acid, drugs, toxins, and vitamins E and K (1, 2, 6).Similar to other P450 2 gene families encoding multipurpose enzymes for the elimination of xenobiotic compounds, family 4 exhibits genetic diversi...

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Section: Resultsmentioning

confidence: 74%

The Crystal Structure of Cytochrome P450 4B1 (CYP4B1) Monooxygenase Complexed with Octane Discloses Several Structural Adaptations for ω-Hydroxylation

Hsu

Baer

Rettie

et al. 2017

Journal of Biological Chemistry

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“…We next examined the identity of the recombinant cytochrome P450 that generates 20-HETE in humans, because conflicting results have been reported on the capability of recombinant P450s to carry out AA hydroxylation (44,45). The predominant catalyst of AA -hydroxylation in human tissues was initially identified as CYP4F2 (46,47).…”

Section: Eet and Aa Metabolismmentioning

confidence: 99%

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

Quéré

Plée-Gautier

Potin

et al. 2004

Journal of Lipid Research

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CYP4F isoforms are involved in the oxidation of important cellular mediators such as leukotriene B 4 (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 K m values of between 27.6 and 175 M. Among recombinant P450 enzymes, CYP4F2 and CYP4F3B catalyzed mainly the -hydroxylation of C18-epoxides with an apparent V max of between 0.84 and 15.0 min ؊ 1 , whereas the apparent V max displayed by CYP4F3A, the isoform found in leukocytes, ranged from 3.0 to 21.2 min ؊ 1 . The rate of -hydroxylation by CYP4A11 was experimentally found to be between 0.3 and 2.7 min ؊ 1 . CYP4F2 and CYP4F3 exhibited preferences for -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 -hydroxylated by liver microsomes and by CYP4F2 and CYP4F3. These data support the hypothesis that the human CYP4F subfamily is involved in the -hydroxylation of fatty acid epoxides. These findings demonstrate that another pathway besides conversion to vicinal diol or chain shortening by ␤ -oxidation exists for fatty acid epoxide inactivation. -Le Quéré, V., E. Plée-Gautier, P. Potin, S. Madec, and J-P. Salaün. Human CYP4F3s are the main catalysts in the oxidation of fatty acid epoxides.

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“…In humans, the predominant -hydroxylases in liver and kidney are microsomal P450s 4A11, 4F2, and 4F3B (4). The -hydroxylation of saturated fatty acids is generally associated with P450 4A enzymes in non-human species, and human P450 4A11 catalyzes the -hydroxylation of lauric acid Ͼ palmitic acid Ͼ arachidonic acid (5). Selective disruption of the murine Cyp4a10 (6) and Cyp4a14 (7) genes leads to hypertensive phenotypes that are thought to reflect changes in renal 20-HETE production arising through distinct mechanisms.…”

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confidence: 99%

Regulation of Human Cytochrome P450 4F2 Expression by Sterol Regulatory Element-binding Protein and Lovastatin

Hsu

Savas

Griffin

et al. 2007

Journal of Biological Chemistry

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The capacity to oxidize the terminal carbon of aliphatic chains is a highly conserved enzymatic activity of cytochrome P450s in plant and animal species. In mammals, the -hydroxylation of fatty acids provides a means to remove potentially toxic excess concentrations of free fatty acids. This is the first step in the formation of dicarboxylic acids that can be more readily excreted or are further degraded by peroxisomal ␤-oxidation. The -hydroxylases also provide pathways for the degradation of signaling molecules such as prostanoids and leukotrienes. On the other hand, -hydroxylation of arachidonic acid forms 20-hydroxyeicosatetraenoic acid (20-HETE), 2 which has been implicated in the regulation of vascular tone and blood pressure (1-3).Cytochrome P450 4F2 (P450 4F2) is a fatty acid -hydroxylase that is expressed in human liver and kidney and contributes to free fatty acid catabolism and the conversion of arachidonic acid to 20-HETE. In humans, the predominant -hydroxylases in liver and kidney are microsomal P450s 4A11, 4F2, and 4F3B (4). The -hydroxylation of saturated fatty acids is generally associated with P450 4A enzymes in non-human species, and human P450 4A11 catalyzes the -hydroxylation of lauric acid Ͼ palmitic acid Ͼ arachidonic acid (5). Selective disruption of the murine Cyp4a10 (6) and Cyp4a14 (7) genes leads to hypertensive phenotypes that are thought to reflect changes in renal 20-HETE production arising through distinct mechanisms. Interestingly, an allelic variant encodes a human CYP4A11 with reduced catalytic activity that is associated with an increased risk for hypertension in studies of three independent cohorts (8, 9). P450s 4F2 and 4F3B are highly similar in amino acid sequence and display overlapping catalytic activity profiles. Both enzymes efficiently oxidize arachidonic acid (10). An alternative transcript of the CYP4F3 gene, 4F3A, arises from differential promoter use leading to the incorporation of an alternative exon that alters substrate preferences to favor the -hydroxylation of leukotrienes (11). P450 4F3A is predominantly expressed in human leukocytes (12). Antibody inhibition experiments (13, 14) suggest that P450 4F2/4F3B in human liver and kidney microsomes contribute ϳ66% to the formation of 20-HETE. Recently, P450s 4F2 and 4F3B were found to -hydroxylate very long chain saturated fatty acids (15) as well as phytanic acid (16). Our preliminary experiments using com-* This work was supported by National Institutes of Health Grant HD004445(to E. F. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

show abstract

Characterization of a cDNA encoding a human kidney, cytochrome 4A fatty acid ω-hydroxylase and the cognate enzyme expressed in Escherichia coli

Cited by 71 publications

References 30 publications

The Crystal Structure of Cytochrome P450 4B1 (CYP4B1) Monooxygenase Complexed with Octane Discloses Several Structural Adaptations for ω-Hydroxylation

The Crystal Structure of Cytochrome P450 4B1 (CYP4B1) Monooxygenase Complexed with Octane Discloses Several Structural Adaptations for ω-Hydroxylation

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

Regulation of Human Cytochrome P450 4F2 Expression by Sterol Regulatory Element-binding Protein and Lovastatin

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