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

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Abstract
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-45016.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-45016.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-45016.alpha. family. There was 94% and 87% nucleotide and amino acid sequence identity, respectively, between P-450cb and C-P-45016.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-P45016.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-45016.alpha.. The results indicated that a broad steroid substrate specificity with high regio- and stereoselectivity at that position was a characteristic of C-P45016.alpha.. We constructed and expressed chimeras between the two P-450s and found that the presence of about two-thirds of the C-P45016.alpha. molecule from its C-terminus was necessary for the chimeric cytochrome to maintain steroid 16.alpha.-hydroxylase activity.

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