Isolation and characterization of a new rat P450 (CYP3A18) cDNA encoding P450_6β-2 catalyzing testosterone 6β- and 16α- hydroxylations

A cytochrome P450 cDNA, encoding a new form of CYP3A protein, was isolated from a liver cDNA library of a male rat using anti-P450_6β-1and anti-P450_6β-2 antibodies and the CYP3A2 cDNA. The cDNA (CYP3A18 cDNA) consisted of 1987 nucleotides, in which were contained an open reading frame of 1491 bp (corresponding to 497 amino acids), 5′-(59bp) and 3′-noncoding regions (437 bp). The deduced amino acid sequence of CYP3A18 cDNA was completely identical in the first 27 N-terminal residues of P450_6β-2 previously isolated by us (Nagata et al. J Biochem 1990: 107, 718-725) from livers of rats treated with dexamethasone, and also shared higher extents of similarity with hamster CYP3A10 (78.5%) than with rat CYP3As previously sequenced (66.3-69.3%). Northern blot analyses indicated a male-dominant expression of this new CYP3A mRNA and enhanced expression in dexamethasone-or pregnenolone-16α-carbonitrile (PCN)-treated, but not phenobarbital- or 3-methylcholanthrene-treated rats. Expressed CYP3A18 protein in COS-1 cells migrated at a position identical to that of purified P450_6β-2 on sodium dodecyl sulfate-acrylamide gel electrophoresis and catalyzed 16β- and 6α-hydroxylations of testosterone. In contrast to CYP3A1 and CYP3A2, cytochrome b₅ was not essential for maximal catalytic activities of recombinant CYP3A18 protein. These results, together with ontogenic profiles of CYP3A18 mRNA and P450_6β-2 protein, indicate that the newly isolated CYP3A18 cDNA encodes P450_6β-2 in rat liver.