title: Cloning and functional expression of a novel marmoset cytochrome P450 2D enzyme, CYP2D30: comparison with the known marmoset CYP2D19.
abstract: 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 > or = 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.
authors.affiliation: Laboratory of Health Chemistry, Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Hichiya
authors.forename: Shino
authors.initials: S
authors.lastname: Kuramoto
authors.forename: Shigeo
authors.initials: S
authors.lastname: Yamamoto
authors.forename: Sumio
authors.initials: S
authors.lastname: Shinoda
authors.forename: Nobumitsu
authors.initials: N
authors.lastname: Hanioka
authors.forename: Shizuo
authors.initials: S
authors.lastname: Narimatsu
authors.forename: Kazuo
authors.initials: K
authors.lastname: Asaoka
authors.forename: Atsuro
authors.initials: A
authors.lastname: Miyata
authors.forename: Shinichi
authors.initials: S
authors.lastname: Iwata
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nomoto
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Satoh
authors.forename: Kimio
authors.initials: K
authors.lastname: Kiryu
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Ueda
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Naito
authors.forename: Geoffrey T
authors.initials: GT
authors.lastname: Tucker
authors.forename: S Wynne
authors.initials: SW
authors.lastname: Ellis
mesh_terms: D000429:Alcohol Oxidoreductases; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002144:Callithrix; D003001:Cloning, Molecular; D000072467:Cytochrome P450 Family 2; D018076:DNA, Complementary / Q000032:analysis; D005260:Female; D015870:Gene Expression; D006801:Humans; D006900:Hydroxylation; D008099:Liver / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D011802:Quinidine / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D000906:Antibodies; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; D001189:Aryl Hydrocarbon Hydroxylases; C504568:Cyp2d1 protein, rat; D000072467:Cytochrome P450 Family 2; C105144:cytochrome P-450 CYP2D19 (marmoset); C487732:cytochrome P-450 CYP2D30 (marmoset); D011802:Quinidine