Identification, functional characterization, and regulation of a new cytochrome P450 subfamily, the CYP2Ns

Marjorie F Oleksiak, Shu Wu, Carol Parker, Sibel I. Karchner, John J. Stegeman, Darryl C. Zeldin

Research output: Contribution to journalArticle

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Abstract

The screening of liver and heart cDNA libraries from the teleost Fundulus heteroclitus with degenerate oligonucleotide probes to conserved α- helical regions in mammalian P450s resulted in the identification of two cDNAs that together represent a novel P450 subfamily, the CYP2Ns. Northern analysis demonstrated that CYP2N1 transcripts are most abundant in liver and intestine, whereas CYP2N2 mRNAs are most abundant in heart and brain. CYP2N1 and CYP2N2 proteins were co-expressed with NADPH-cytochrome P450 oxidoreductase in Sf9 insect cells, and their ability to metabolize arachidonic acid and xenobiotic substrates was examined. Both CYP2N1 and CYP2N2 metabolize arachidonic acid to epoxyeicosatrienoic acids. Epoxidation is highly regio- and enantioselective with preferential formation of (8R,9S)- epoxyeicosatrienoic acid (optical purities are 91 and 90% for CYP2N1 and CYP2N2, respectively) and (11R,12S)-epoxyeicosatrienoic acid (optical purities are 92 and 70% for CYP2N1 and CYP2N2, respectively). CYP2N1 and CYP2N2 also catalyze the formation of a variety of hydroxyeicosatetraenoic acids. Both P450s have benzphetamine N-demethylase activities but show minimal alkoxyresorufin O-dealkylase activities. To investigate factors affecting CYP2N expression in vivo, CYP2N transcripts were examined following starvation and/or treatment with 12-O-tetradecanoyl phorbol-13-acetate. Intestinal CYP2N1 mRNAs decrease in starved and/or phorbol ester-treated fish, whereas intestinal CYP2N2 transcripts decrease only following phorbol ester treatment. Interestingly, cardiac CYP2N2 expression decreases following phorbol ester treatment but increases following starvation. These results demonstrate that members of this novel P450 subfamily encode early vertebrate forms of arachidonic acid catalysts that are widely expressed and are regulated by environmental factors. Given the wealth of information on the functional role of P450-derived arachidonate metabolites in mammals, we postulate that CYP2N1 and CYP2N2 products have similar biological functions in early vertebrates. The identity of the mammalian orthologue(s) of the CYP2Ns remains unknown.

Original languageEnglish
Pages (from-to)2312-2321
Number of pages10
JournalJournal of Biological Chemistry
Volume275
Issue number4
DOIs
StatePublished - Jan 28 2000
Externally publishedYes

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Cytochrome P-450 Enzyme System
Phorbol Esters
Arachidonic Acid
Starvation
Liver
Acids
Vertebrates
Fundulidae
Sf9 Cells
Hydroxyeicosatetraenoic Acids
cytochrome P-450 CYP2N2 (killifish)
NADPH-Ferrihemoprotein Reductase
Messenger RNA
Mammals
Epoxidation
Oligonucleotide Probes
cytochrome P-450 CYP2N1 (killifish)
Tetradecanoylphorbol Acetate
Xenobiotics
Metabolites

ASJC Scopus subject areas

  • Biochemistry

Cite this

Identification, functional characterization, and regulation of a new cytochrome P450 subfamily, the CYP2Ns. / Oleksiak, Marjorie F; Wu, Shu; Parker, Carol; Karchner, Sibel I.; Stegeman, John J.; Zeldin, Darryl C.

In: Journal of Biological Chemistry, Vol. 275, No. 4, 28.01.2000, p. 2312-2321.

Research output: Contribution to journalArticle

Oleksiak, Marjorie F ; Wu, Shu ; Parker, Carol ; Karchner, Sibel I. ; Stegeman, John J. ; Zeldin, Darryl C. / Identification, functional characterization, and regulation of a new cytochrome P450 subfamily, the CYP2Ns. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 4. pp. 2312-2321.
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