Structure of a Gene and cDNA of a Major Constitutive Form of Testosterone 6β-Hydroxylase (P450/6βA) Encoding CYP3A2: Comparison of the cDNA with P450PCN2

• Published in: Archives of biochemistry and biophysics Vol. 314; no. 2; pp. 351 - 359
• Main Authors: Miyata, M., Nagata, K., Shimada, M., Yamazoe, Y., Kato, R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.1994
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Cell Line; Cercopithecus aethiops; Cytochrome P-450 Enzyme System - biosynthesis; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; DNA Primers; DNA, Complementary - analysis; Genetic Variation; Genomic Library; Liver - enzymology; Male; Molecular Sequence Data; Polymerase Chain Reaction; Rats; Sequence Homology, Nucleic Acid; Steroid Hydroxylases - biosynthesis; Steroid Hydroxylases - genetics; Steroid Hydroxylases - metabolism; Transfection
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1006/abbi.1994.1453

P450/6βA gene was isolated from a rat genomic library and its encoding protein was characterized by the expression of the corresponding cDNA in COS-1 cells. Between exon regions of P450/6βA gene and P450PCN2 cDNA (F. J. Gonzalez, B.-J. Song, and J. P. Hardwick (1986) Mol. Cell. Biol. 6, 2969-2976), 12 nucleotide differences were observed, involving two amino acid changes, His → Asp [429] and Asp → Gly [445], respectively. A cDNA (6βA cDNA), whose nucleotide sequence was completely identical with the corresponding exon of P450/6βA gene, was isolated from a rat cDNA library. Northern blotting using specific oligonucleotide probes showed that 6β-A mRNA, but not P450PCN2 mRNA, was a major form in livers of the male rats. 6β-A protein expressed in COS-1 cells (at about 0.1 to 0.3% of total microsomal protein) catalyzed testosterone 6β-hydroxylation. The reaction was enhanced by the addition of NADPH-P450 reductase (2.5-fold) and by the simultaneous addition of cytochrome b5 and NADPH-P450 reductase (13.7-fold). Catalytic properties of 6β-A for typical CYP3A substrates were consistent with those of purified rat P4506β-1 and P4506β-3, corresponding, respectively, to CYP3A2 or the variant form (K. Nagata, F. J. Gonzalez, Y. Yamazoe, and R. Kato (1990) J. Biochem. 107, 718-725). Apparent Michaelis constants (Km) of 6β-A for testosterone 6β-hydroxylation and a O-dealkylation ratio of propoxycoumarin to pentoxycoumarin, however, showed higher degrees of similarity to those of purified P4506β-3 than those of P4506β-1.