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

The screening of liver and heart cDNA libraries from the teleost Fundulus h eteroclitus with degenerate oligonucleotide probes to conserved alpha-helic al regions in mammalian P450s resulted in the identification of two cDNAs t hat together represent a novel P450 subfamily, the CYP2Ns. Northern analysi s demonstrated that CYP2N1 transcripts are most abundant in liver and intes tine, whereas CYP2N2 mRNAs are most abundant in heart and brain. CYP2N1 and CYP2N2 proteins were co-expressed with NADPH cytochrome P450 oxidoreductas e in Sf9 insect cells, and their ability to metabolize arachidonic acid and xenobiotic substrates was examined. Both CYP2N1. and CYP2N2 metabolize ara chidonic acid to epoxyeicosatrienoic acids. Epoxidation is highly regio- an d enantioselective with preferential formation of (8R,9S)-epoxyeicosatrieno ic acid (optical purities are 91 and 90% for CYP2N1 and CYP2N2, respectivel y) and (11R,12S)-epoxyeicosatrienoic acid (optical purities are 92 and 70% for CYP2N1 and CYP2N2, respectively). CYP2N1 and CYP2N2 also catalyze the f ormation of a variety of hydroxyeicosatetraenoic acids. Both P450s have ben zphetamine N-demethylase activities but show minimal alkoxyresorufin O-deal kylase activities. To investigate factors affecting CYP2N expression in viv o, CYP2N transcripts were examined following starvation and/or treatment wi th 12-O-tetradecanoyl phorbol-13-acetate. Intestinal CYP2N1 mRNAs decrease in starved and/or phorbol ester-treated fish, whereas intestinal CYP2N2 tra nscripts decrease only following phorbol ester treatment. Interestingly, ca rdiac CYP2N2 expression decreases following phorbol ester treatment but inc reases following starvation. These results demonstrate that members of this novel P450 subfamily encode early vertebrate forms of arachidonic acid cat alysts that are widely expressed and are regulated by environmental factors . Given the wealth of information on the functional role of P450-derived ar achidonate metabolites in mammals, we postulate that CYP2N1 and CYP2N2 prod ucts have similar biological functions in early vertebrates. The identity o f the mammalian orthologue(s) of the CYP2Ns remains unknown.