Complementary DNA cloning and characterization of cytochrome P450 2D29 from Japanese monkey liver

Abstract

A cDNA was cloned from Japanese monkey liver mRNA by reverse transcriptase–polymerase chain reaction (RT–PCR) using oligonucleotide primers based on the marmoset cytochrome P450 2D19 (CYP2D19) nucleotide sequence. The full-length cDNA encoded a 497 amino acid protein (designated CYP2D29) that is 96, 91, and 88% homologous to human CYP2D6, cynomolgus monkey CYP2D17, and marmoset monkey CYP2D19, respectively. Yeast cells ( Saccharomyces cerevisiae AH-22 strain) transfected with pGYR1 vectors containing the CYP2D29 cDNA were cultured, and microsomal fractions were obtained. Reduced carbon monoxide-difference spectra and western blot analysis using polyclonal antibodies raised against rat CYP2D2 demonstrated that in yeast cell microsomal fractions, the level of CYP2D29 holoenzyme was similar to that of CYP2D6 holoenzyme. However, western blot analysis indicated that the level of CYP2D29 in Japanese monkey liver microsomes might be much higher than that of CYP2D6 in human liver microsomes. Japanese monkey liver microsomes exhibited much higher activities than did human liver microsomes, expressed as nmol/min/mg protein, for debrisoquine (DB) 4-hydroxylation and bufuralol (BF) 1″-hydroxylation (typical reactions catalyzed by CYP2D6), whereas recombinant CYP2D29 activity, expressed as nmol/min/nmol CYP, was similar to that of CYP2D6 for DB and BF hydroxylation. In kinetic analyses, the K m value of CYP2D29 for DB 4-hydroxylation was much lower than that of Japanese monkey liver microsomes, whereas the K m value of CYP2D6 for DB 4-hydroxylation was similar to that of human liver microsomes. In contrast, K m values for BF 1″-hydroxylation were similar for Japanese monkey and human liver microsomes and yeast cell microsomal fractions expressing recombinant CYP2D29 or CYP2D6. These results suggest that the properties of Japanese monkey CYP2D29 are similar to those of human CYP2D6, but their populations and/or some other factors in liver microsomes may cause the difference in microsomal DB 4-hydroxylase activities between Japanese monkeys and humans.