Cloning and expression of CYP2F3, a cytochrome P450 that bioactivates the selective pneumotoxins 3-methylindole and naphthalene.
Members of the CYP2F gene subfamily are selectively expressed in lung tissues and have been implicated as important catalysts in the formation of reactive intermediates from several pneumotoxic chemicals. Human CYP2F1 bioactivates 3-methylindole (3MI), while mouse CYP2F2 bioactivates naphthalene. Although 3MI is a potent pneumotoxin in ruminants and rodents, the participation of cytochrome P450s from the 2F subfamily in 3MI bioactivation has not been fully defined. To test the hypothesis that a goat lung 2F homologue uniquely catalyzes the dehydrogenation of 3MI to the putative electrophile 3-methylene-indolenine, the CYP2F3 cDNA was cloned from a goat lung cDNA library and expressed in Escherichia coli. The predicted amino acid sequence of CYP2F3 possessed 82% identity to both human CYP2F1 and mouse CYP2F2. CYP2F3 was mutated at the 5' end, expressed in E. coli, and shown to have a molecular mass of 50 kDa. The reconstituted enzyme uniquely catalyzed only the dehydrogenation of 3MI to form 3-methylene-indolenine, an electrophilic intermediate, without detectable formation of other products, thus demonstrating highly unusual selectivity for dehydrogenation rather than hydroxylation of a substrate. Immunoinhibition studies demonstrated that about 20% of the production of the intermediate in goat lung microsomal samples was produced by CYP2F3. The CYP2F3 enzyme had a specific activity that was similar to that of human cDNA-expressed CYP2F1. CYP2F3 also stereoselectively catalyzed the formation of the 1R,2S-oxide from naphthalene; this stereoisomer is the putative pneumotoxin. The enzyme, however, lacked catalytic activity with other common P450 substrates including 7-ethoxycoumarin, a substrate for CYP2F1, indicating that the substrate selectivity of CYP2F3 appears to be high.
Wang
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Department of Pharmacology and Toxicology, University of Utah, Salt Lake City 84112, USA.
Lanza
D L
DL
Yost
G S
GS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001711:Biotransformation; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004926:Escherichia coli; D006041:Goats; D006801:Humans; D008168:Lung / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D008297:Male; D051379:Mice; D008861:Microsomes / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009281:Naphthalenes / Q000493:pharmacokinetics* / Q000633:toxicity; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012862:Skatole / Q000493:pharmacokinetics* / Q000633:toxicity; D013379:Substrate Specificity
D009281:Naphthalenes; D011994:Recombinant Proteins; C031721:naphthalene; D003577:Cytochrome P-450 Enzyme System; D012862:Skatole; C110333:Cyp2f2 protein, mouse; C110332:CYP2F1 protein, human; D000072467:Cytochrome P450 Family 2; C110331:cytochrome P-450 CYP2F3 (goat)