Cloning and expression of two novel pig liver and kidney fatty acid hydroxylases [cytochrome P450 (CYP)4A24 and CYP4A25]

Lundell, Kerstin

doi:10.1042/bj3630297

Cited by 13 publications

(8 citation statements)

References 20 publications

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“…Protein concentration was estimated by the Bradford method using γ‐globulin as standard (Bradford 1976). HPLC analysis of metabolites of [ 14 C]lauric acid was performed as described (Lundell 2002).…”

Section: Methodsmentioning

confidence: 99%

Expression of CYP4F12 in Gastrointestinal and Urogenital Epithelia*

Stark

Schauer

Sahlén

et al. 2004

Basic Clin Pharma Tox

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Cytochrome P450 4F12 (CYP4F12) was originally cloned from human liver and small intestine. CYP4F12 can oxidize arachidonic acid, two stable prostaglandin H2 analogues, and an antihistamine, ebastine, but the tissue distribution and catalytic properties of CYP4F12 have not been fully investigated. An antipeptide polyclonal antibody was raised against the C-terminal of CYP4F12 (PLNVGLQ), evaluated by Western blot analysis and used for immunohistological analysis of 50 human tissues. Western blot analysis of recombinant CYP4F12, expressed in yeast, and microsomal proteins from adult and foetal liver, kidney, placenta at term, seminal vesicles, the prostate gland and purified prostasomes showed that the polyclonal antibody detected a protein of the expected size, approximately 60 kDa. CYP4F12 mRNA could be detected in seminal vesicles and prostate gland by reverse transcription-PCR. Prominent CYP4F12 immunoreactivity occurred, inter alia, in the epithelial cells of the gastrointestinal tract (stomach, small intestine, and colon), collecting tubules, transitional epithelium, ovarian follicles, the endothelium of microvessels of placental villi (first trimester), and epidermis. We screened recombinant CYP4F12 for catalytic activity. Arachidonic acid (20:4n-6) was hydroxylated at C18 and laurate at C11, but significant amounts of metabolites of 18:2n-6, 20:3n-9, 20:5n-3, 22:5n-6, and some prostaglandins could not be detected. We conclude that CYP4F12 is widely distributed in gastrointestinal and urogenital epithelia and exhibits a narrow substrate specificity.

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Section: Methodsmentioning

confidence: 99%

Expression of CYP4F12 in Gastrointestinal and Urogenital Epithelia*

Stark

Schauer

Sahlén

et al. 2004

Basic Clin Pharma Tox

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“…The primary structure of the porcine CYP4A21, acting as a 6 -hydroxylase, showed about 75% identity with known members of the CYP4A subfamily in rabbit and man, and 97% and 94% overall identity to two fatty acid hydroxylases expressed in pig, CYP4A24 and 25 [112]. A peculiarity was that the conserved signature sequence previously found among members of the CYP4A and 4B enzymes [108] contained deviations as shown in Fig.…”

Section: Porcine Taurochenodeoxycholic Acid 6 -Hydroxylase Cyp-4a21mentioning

confidence: 96%

Species-Specific and Age-Dependent Bile Acid Composition: Aspects on CYP8B and CYP4A Subfamilies in Bile Acid Biosynthesis

Lundell

Wikvall²

2008

CDM

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The present review aims to give an overview of the cytochrome P450 8B (CYP8B) and cytochrome P450 4A (CYP4A) subfamilies in relation to biosynthesis of bile acids, in particular trihydroxy bile acids. Trihydroxy bile acids are basically required in most species and have an impact on cholesterol and lipid metabolism. The primary trihydroxy bile acid in most mammals is cholic acid. Some species produce other important trihydroxy bile acids, for example the adult pig which produce hyocholic acid instead of cholic acid. The position of the third hydroxyl group in cholic acid and hyocholic acid, 12alpha or 6alpha position, respectively, has a profound effect on the hydrophilic-hydrophobic property of the trihydroxy bile acids. The CYP8B subfamily is required for introduction of the 12alpha-hydroxyl group in cholic acid biosynthesis. The enzyme responsible for 6alpha-hydroxylation in hyocholic acid biosynthesis, however, varies among species. This review will discuss, in particular, porcine members of the CYP8B and CYP4A subfamilies because interesting findings regarding members of these subfamilies have recently been recognized in this species. CYP8B1 was for a long time believed to be absent in the pig but was recently found to be expressed in fetal pig liver. The enzyme catalyzing the 6alpha-hydroxylation in hyocholic acid biosynthesis in pig was found to be an atypical member of the CYP4A subfamily, denoted CYP4A21. The review presents bile acid biosynthesis in view of these findings and discusses physiochemical properties and developmental-dependent aspects related cholic acid and hyocholic acid biosynthesis.

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“…The catalytic specificities of the rat enzymes CYP4A1, CYP4A2, CYP4A3, and CYP4A8 have been investigated [20], as have the fatty acid ω-hydroxylase activities of CYP4 enzymes from mice [21], rabbits [22], pigs [23], and other mammals. In plants CYP76B9 [24], CYP78A1 [25], CYP86A1 [26], CYP86A8 [27], CYP86A22 [28], CYP94A1 [29], CYP94A2 [30], CYP94A5 [31], CYP94C1 [32], CYP96A1 [33], CYP96A2 [33], and CYP97B3 [33] have been shown to ω-hydroxylate fatty acids, although the substrate for the last enzyme is the thioester CoA derivative rather than the free acid.…”

Section: Enzymes That Predominantly Perform ω-Hydroxylationmentioning

confidence: 99%

Structural control of cytochrome P450-catalyzed ω-hydroxylation

Johnston

Ouellet

Podust

et al. 2011

Archives of Biochemistry and Biophysics

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The regiospecific or preferential ω-hydroxylation of hydrocarbon chains is thermodynamically disfavored because the ease of C-H bond hydroxylation depends on the bond strength, and the primary C-H bond of a terminal methyl group is stronger than the secondary or tertiary C-H bond adjacent to it. The hydroxylation reaction will therefore occur primarily at the adjacent secondary or tertiary C-H bond unless the protein structure specifically enforces primary C-H bond oxidation. Here we review the classes of enzymes that catalyze ω-hydroxylation and our current understanding of the structural features that promote the ω-hydroxylation of unbranched and methyl-branched hydrocarbon chains. The evidence indicates that steric constraints are used to favor reaction at the ω-site rather than at the more reactive (ω-1)-site.Keywords cytochrome P450; ω-hydroxylation; CYP4 family; carbon hydroxylation; fatty acids; branchedchain hydrocarbon acids; cholesterol degradation Hydrocarbon ω-hydroxylationIn chemical terms, the regio-and stereoselective oxidation of unactivated hydrocarbon C-H bonds to the corresponding hydroxy (C-OH) products is the most difficult reaction catalyzed by cytochrome P450 enzymes. This substrate hydroxylation reaction is mediated by the "Compound I"-like ferryl species formed during the catalytic turnover of P450 enzymes. The Fe(IV) heme iron atom in this ferryl species is paired with a radical cation delocalized over the heme porphyrin ring, so the enzyme is two oxidation equivalents higher than the resting enzyme. The formation of this oxidizing intermediate via the catalytic cycle in Fig. 1 has been extensively reviewed and is therefore not discussed here [1].The hydroxylation of a hydrocarbon chain is initiated by abstraction of the hydrogen atom of the C-H bond by the ferryl oxygen atom, yielding a transient carbon radical coupled to an Fe(IV)-OH catalytic intermediate. Rebound collapse of a hydroxyl radical equivalent from the iron with the substrate radical produces the alcohol and returns the enzyme to the resting © 2010 Elsevier Inc. All rights reserved.Corresponding author: Paul R. Ortiz de Montellano, University of California, San Francisco, San Francisco, CA 94158-2517, Tel: +1 (415) 476-2903, Fax: +1 (415) 502-4728, ortiz@cgl.ucsf.edu . Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Fig. 2). Computational and experimental results indicate that the ease of oxidation of any given C-H bond is related to its bond strength; i.e., to the energy required to homolytically break the C-H into a carbon radical and a hydrogen radical (C-H -> C ....

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Cloning and expression of two novel pig liver and kidney fatty acid hydroxylases [cytochrome P450 (CYP)4A24 and CYP4A25]

Cited by 13 publications

References 20 publications

Expression of CYP4F12 in Gastrointestinal and Urogenital Epithelia*

Expression of CYP4F12 in Gastrointestinal and Urogenital Epithelia*

Species-Specific and Age-Dependent Bile Acid Composition: Aspects on CYP8B and CYP4A Subfamilies in Bile Acid Biosynthesis

Structural control of cytochrome P450-catalyzed ω-hydroxylation

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