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

Hichiya, Hiroyuki; Takemi, Chie; Tsuzuki, Daisuke; Yamamoto, Satoshi; Asaoka, Kazuo; Suzuki, Satoshi; Satoh, Tetsuo; Shinoda, Sumió; Kataoka, Hiroyuki; Narimatsu, Shizuo

doi:10.1016/s0006-2952(02)01287-x

Cited by 23 publications

(18 citation statements)

References 26 publications

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“…Assay of Drug Oxidation Activities ---DB 4-hydroxylase activity was measured by the HPLC method reported previously. 6) Briefly, a 500-µl incubation mixture in an Eppendorf-type tube (1.5 ml) contained 10 pmol of CYP, 5 mM G-6-P, 1 IU of G-6-P dehydrogenase, 5 mM MgCl 2 , 0.1 mM EDTA, 0.5 mM NADPH and 100 µM DB in 100 mM potassium phosphate buffer (pH 7.4). After a 5-min preincubation at 37°C, the reaction was started by adding NADPH and continued for 5 min.…”

Section: )mentioning

confidence: 99%

“…6) Briefly, a 500 µl incubation mixture in a brown glass conical tube (10 ml) with a glass stopper contained 10 pmol CYP, 5 mM G-6-P, 1 IU of G-6-P dehydrogenase, 5 mM MgCl 2 , 0.1 mM EDTA, 0.5 mM NADPH and 100 µM BF racemate in 100 mM potassium phosphate buffer (pH 7.4). Manipulations hereafter were the same as those employed in the assay of BTL 4-hydroxylation except for using propranolol racemate (100 nmol) instead of procainamide as an internal standard.…”

Section: )mentioning

confidence: 99%

“…[4][5][6] In our previous project, CYP2D2 (P450BTL) was purified employing bunitrolol (BTL) 4-hydroxylase as the index of CYP2D2 functions. 4) BTL is a β-adrenoceptor blocking agent, and rat CYP2D2, 4) as well as human CYP2D6, 7) catalyzes BTL 4-hydroxylation with relatively low Km values.…”

Section: Introductionmentioning

confidence: 99%

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Changes in the Enzymatic Properties of CYP2D6 by the Substitution of Phenylalanine at Position 120 by Alanine

Masuda

Hashimoto

Tamagake

et al. 2004

JOURNAL OF HEALTH SCIENCE

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The functional roles of phenylalanine at position 120 (Phe-120) in the oxidation of bunitrolol (BTL), debrisoquine (DB) and bufuralol (BF) by cytochrome P450 2D6 (CYP2D6) were examined using a yeast cell expression system (Saccharomyces cerevisiae AH-22 strain). The substitution of Phe-120 by alanine markedly increased the activities of enantiomeric BTL 4-hydroxylase and DB 4-hydroxylase, whereas it did not remarkably affect BF 1′′-hydroxylase activities. Kinetic studies revealed that the substitution of Phe-120 by alanine increased the Km and Vmax values for enantiomeric BTL 4-hydroxylation, but increased only the Vmax value for DB 4-hydroxylation without changing the Km value. Km and Vmax values for BF 1′′-hydroxylation were similar between the mutant and the wild-type. The dissociation constants of the mutant calculated from the binding spectra for BTL enantiomers were higher than those of the wild-type, suggesting that the substitution of Phe-120 by alanine decreased the affinity of CYP2D6 for BTL enantiomers. These results indicate that Phe-120 has an important role in the oxidation of substrates by CYP2D6.

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confidence: 99%

Section: )mentioning

confidence: 99%

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Changes in the Enzymatic Properties of CYP2D6 by the Substitution of Phenylalanine at Position 120 by Alanine

Masuda

Hashimoto

Tamagake

et al. 2004

JOURNAL OF HEALTH SCIENCE

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“…The former two are used routinely in preclinical studies. Cynomolgus monkey CYP2D17 (Mankowski et al, 1999) and Japanese monkey CYP2D29 (Hichiya et al, 2002) have been identified and characterized in macaques. Rhesus monkey CYP2D42 cDNA, nearly 98% homologous to cynomolgus monkey CYP2D17 cDNA, has been isolated and its sequence can be found in GenBank.…”

Section: Introductionmentioning

confidence: 99%

“…Deduced amino acid sequences of these CYP2D cDNAs share high sequence identity (93-96%) with human CYP2D6. Cynomolgus monkey CYP2D17 and Japanese monkey CYP2D29 metabolize typical human CYP2D6 substrates; CYP2D17 metabolizes bufuralol and dextromethorphan, whereas CYP2D29 metabolizes bufuralol and debrisoquine (Mankowski et al, 1999;Hichiya et al, 2002). Moreover, cynomolgus monkey CYP2D17 and Japanese monkey CYP2D29 enzymatic activities are inhibited by the CYP2D6 inhibitor quinidine, indicating that these macaque CYP2Ds have functional characteristics similar to those of human CYP2D6.…”

Section: Introductionmentioning

confidence: 99%

Cynomolgus Monkey CYP2D44 Newly Identified in Liver, Metabolizes Bufuralol, and Dextromethorphan

Uno

Uehara²,

Kohara³

et al. 2010

Drug Metab Dispos

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ABSTRACT:The cynomolgus monkey is used in drug metabolism studies, because of its evolutionary closeness to human, including cytochrome P450. Cynomolgus monkey CYP2D17, highly homologous to human CYP2D6, has been identified and characterized. Here, we report characterization of another CYP2D, CYP2D44, identified in cynomolgus monkey liver. The CYP2D44 cDNA contained an open reading frame of 497 amino acids sharing high sequence identity (87-93%) with other primate CYP2Ds. CYP2D44 mRNA was predominantly expressed in liver, similar to CYP2D17 mRNA. CYP2D17 and CYP2D44 form a gene cluster in the genome, similar to human CYP2Ds. Metabolic assays of the CYP2D17 and CYP2D44 proteins heterologously expressed in Escherichia coli indicated that CYP2D44 metabolized human CYP2D6 substrates, bufuralol and dextromethorphan (bufuralol 1-hydroxylation and dextromethorphan O-demethylation) but to a lesser extent than CYP2D17. Kinetic analysis of dextromethorphan metabolism indicated that the apparent K m and V max of CYP2D17 and CYP2D44 catalyzed O-demethylation were similar, and, the V max values of CYP2D17 and CYP2D44 catalyzed Ndemethylation (which human CYP2D6 catalyzes much less effectively) were similar, but the apparent K m of the CYP2D44 reaction was higher. Western blot analysis showed that CYP2D proteins were expressed in cynomolgus and rhesus monkey liver as well as in human and marmoset liver. Similar to CYP2D6, CYP2D44 copy number varied among the eight cynomolgus monkeys and four rhesus monkeys used in this study. These results indicated that CYP2D44, together with CYP2D17, had functional characteristics similar to those of human CYP2D6 but measurably differed in dextromethorphan N-demethylation, suggesting its importance for CYP2D-dependent drug metabolism in macaque.

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Change in enantioselectivity in bufuralol 1?-hydroxylation by the substitution of phenylalanine-120 by alanine in cytochrome P450 2D6

et al. 2004

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The functional roles of phenylalanine at position 120 in drug oxidation by cytochrome P450 2D6 (CYP2D6) were examined using a yeast cell expression system and bufuralol (BF) enantiomers as a chiral substrate. Two mutated cDNAs, one encoding a CYP2D6 mutant having alanine instead of Phe-120 (F120A) and another encoding a mutant having alanine instead of Glu-222 (E222A), were prepared by site-directed mutagenesis and transformed into yeast cells via pGYRI vectors. The enantiomeric BF 1''-hydroxylase activities of the mutants were compared with those of the wild type. When enantiomeric BF 1''-hydroxylase activities at a substrate concentration of 100 microM were compared, the CYP2D6 wild type showed substrate enantioselectivity of (R-BF >> S-BF) and the F120A mutant exhibited substrate enantioselectivity of (R-BF < or = S-BF), whereas the product diastereoselectivity of (1''R-OH-BF << 1''-S-OH-BF) was similar between the wild type and the mutant. The activities of the other mutant (E222A) were much lower than those of the wild type and the F120A mutant, while its substrate enantioselectivity and product diastereoselectivity were the same as those of the wild type. The kinetics demonstrated that apparent K(m) values were similar among the recombinant enzymes, and V(max) values clearly reflected the selectivity described above. These results indicate that Phe-120 has a key role in the enantioselective BF 1''-hydroxylation by CYP2D6.

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

Cited by 23 publications

References 26 publications

Changes in the Enzymatic Properties of CYP2D6 by the Substitution of Phenylalanine at Position 120 by Alanine

Changes in the Enzymatic Properties of CYP2D6 by the Substitution of Phenylalanine at Position 120 by Alanine

Cynomolgus Monkey CYP2D44 Newly Identified in Liver, Metabolizes Bufuralol, and Dextromethorphan

Change in enantioselectivity in bufuralol 1?-hydroxylation by the substitution of phenylalanine-120 by alanine in cytochrome P450 2D6

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