Cloning and functional expression of a novel marmoset cytochrome P450 2D enzyme, CYP2D30: comparison with the known marmoset CYP2D19

• Published in: Biochemical pharmacology 2004-07, Vol.68 (1), p.165-175
• Main Authors: Hichiya, Hiroyuki, Kuramoto, Shino, Yamamoto, Shigeo, Shinoda, Sumio, Hanioka, Nobumitsu, Narimatsu, Shizuo, Asaoka, Kazuo, Miyata, Atsuro, Iwata, Shinichi, Nomoto, Masahiro, Satoh, Tetsuo, Kiryu, Kimio, Ueda, Nobuhiko, Naito, Shinsaku, Tucker, Geoffrey T., Ellis, S.Wynne
• Format: Article
• Language: English
• Subjects: Alcohol Oxidoreductases; Amino Acid Sequence; Animals; Antibodies - pharmacology; Aryl Hydrocarbon Hydroxylases - chemistry; Aryl Hydrocarbon Hydroxylases - genetics; Aryl Hydrocarbon Hydroxylases - metabolism; Base Sequence; Bufuralol; Callithrix; Cloning, Molecular; CYP2D19; CYP2D30; CYP2D6; Cytochrome P450 Family 2; DNA, Complementary - analysis; Female; Gene Expression; Humans; Hydroxylation; Liver - enzymology; Marmoset, debrisoquine; Microsomes, Liver - enzymology; Microsomes, Liver - metabolism; Molecular Sequence Data; Quinidine - pharmacology; Rats; Recombinant Proteins - metabolism; RNA, Messenger - analysis; Sequence Homology, Amino Acid
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/j.bcp.2004.03.018

Using a primer set designed on the cDNA encoding the known marmoset cytochrome P450 2D19 (CYP2D19), a cDNA encoding a novel CYP2D enzyme (CYP2D30) was cloned from the liver of a female marmoset bred at Kyoto University (KYU). In addition, a cDNA encoding CYP2D19 was cloned from the liver of a female marmoset bred at Kagoshima University (KAU). CYP2D30 and CYP2D19 showed homologies of 93.6 and 93.4% in their nucleotide and amino acid sequences, respectively. Reverse transcription polymerase chain reaction (RT-PCR) and digestion with NdeI demonstrated that the KYU-marmoset liver contained mainly mRNA for CYP2D30, while the KAU-marmoset liver contained mainly mRNA for CYP2D19. Marmoset CYP2D30, like human CYP2D6, exhibited high debrisoquine (DB) 4-hydroxylase activity and relatively low DB 5-, 6-, 7- and 8-hydroxylase activities, whereas CYP2D19 lacked DB 4-hydroxylase but exhibited marked 5-, 6-, 7- and 8-hydroxylase activities. The two marmoset recombinant enzymes showed enantioselective bufuralol (BF) 1″-hydroxylase activities, similar to CYP2D6. BF 1″-hydroxylation by CYP2D30 exhibited product-enantioselectivity of (1″ R-OH-BF ⪡ 1″ S-OH-BF), similar to that observed with human CYP2D6, whereas CYP2D19 showed a reversed selectivity of (1″ R-OH-BF ≥ 1″ S-OH-BF). BF 1″-hydroxylation in marmoset liver microsomes from both sources was inhibited by antibodies raised against rat CYP2D1 in a concentration-dependent manner. A known inhibitor of CYP2D6, quinidine, effectively inhibited the BF 1″-hydroxylation activities in liver microsomal fractions prepared from KYU- and KAU-marmosets. These results suggest that CYP2D19 and CYP2D30 proteins can be expressed as functional enzymes in marmoset livers, although it is unresolved whether both enzymes coexist in the same marmoset liver.