Functional characterization of two cytochrome P-450s within the mouse, male-specific steroid 16 alpha-hydroxylase gene family: expression in mammalian cells and chimeric proteins.
Two cDNAs, pc16 alpha-2 and pc16 alpha-25, which encode P-450s from within the mouse, male-specific steroid 16 alpha-hydroxylase (C-P-450(16 alpha)) gene family, were transfected into COS-1 cells in order to study catalytic activities of the expressed P-450s. pc16 alpha-2 was shown previously to encode the growth hormone dependent and androgen-dependent C-P-450(16 alpha) in adult male mice (Wong et al., 1987). The sequence of pc16 alpha-25-encoded P-450 (P-450cb) was identical with gene cb within the C-P-450(16 alpha) family. There was 94% and 87% nucleotide and amino acid sequence identity, respectively, between P-450cb and C-P-450(16 alpha). We expressed both P-450s by transfecting their cDNAs into COS-1 cells and found that steroid 16 alpha-hydroxylase activity was catalyzed by C-P-450(16 alpha) but not by P-450cb. In addition to testosterone, progesterone and estradiol were hydroxylated specifically at the 16 alpha-position by the expressed C-P-450(16 alpha). The results indicated that a broad steroid substrate specificity with high regio- and stereoselectivity at that position was a characteristic of C-P-450(16 alpha). We constructed and expressed chimeras between the two P-450s and found that the presence of about two-thirds of the C-P-450(16 alpha) molecule from its C-terminus was necessary for the chimeric cytochrome to maintain steroid 16 alpha-hydroxylase activity.
Ichikawa
T
T
Pharmacogenetics Section, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Itakura
T
T
Negishi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D002678:Chimera; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000032:analysis; D015245:Deoxyribonuclease BamHI; D015252:Deoxyribonucleases, Type II Site-Specific; D005091:Exons; D005786:Gene Expression Regulation; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D012737:Sex Factors; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase; D015245:Deoxyribonuclease BamHI; C032824:ATCGAT-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific