Cloning of a cDNA encoding a novel marmoset CYP2C enzyme, expression in yeast cells and characterization of its enzymatic functions.
We cloned a cDNA encoding a novel CYP2C enzyme, called P450 M-2C, from a marmoset liver. The deduced amino acid sequence showed high identities to those of human CYP2C8 (87%), CYP2C9 (78%) and CYP2C19 (77%). The P450 M-2C enzyme expressed in yeast cells catalyzed p-methylhydroxylation of only tolbutamide among four substrates tested, paclitaxel as a CYP2C8 substrate, diclofenac and tolbutamide as CYP2C9 substrates and S-mephenytoin as a CYP2C19 substrate. p-Methylhydroxylation of tolbutamide by marmoset liver microsomes showed monophasic kinetics, and the apparent K(m) value (1.2 mM) for the substrate was similar to that of the recombinant P450 M-2C (1.8 mM). Although all of the recombinant human CYP2C8, CYP2C9 and CYP2C19 expressed in yeast cells catalyzed tolbutamide p-methylhydroxylation, the kinetic profile of CYP2C8 was most similar to that of P450 M-2C. Tolbutamide oxidation by the marmoset liver microsomes and the recombinant P450 M-2C was inhibited most effectively by quercetin, a CYP2C8 inhibitor, followed by omeprazole, a CYP2C19 inhibitor, whereas sulfaphenazole, a CYP2C9 inhibitor, was less potent under the conditions used. These results indicate that P450 M-2C is the major tolbutamide p-methylhydroxylase in the marmoset liver.
Narimatsu
Shizuo
S
Laboratory of Health Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan. shizuo@pharm.okayama-u.ac.jp
Torigoe
Fumihiro
F
Tsuneto
Yumi
Y
Saito
Keita
K
Hanioka
Nobumitsu
N
Masuda
Kazufumi
K
Katsu
Takashi
T
Yamamoto
Shigeo
S
Yamano
Shigeru
S
Baba
Takahiko
T
Miyata
Atsuro
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D002144:Callithrix; D003001:Cloning, Molecular; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D006900:Hydroxylation; D008862:Microsomes, Liver / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D016415:Sequence Alignment; D013379:Substrate Specificity
D065607:Cytochrome P-450 Enzyme Inhibitors; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; C400322:cytochrome P-450 CYP2C subfamily; D003577:Cytochrome P-450 Enzyme System