Cynomolgus monkey cytochrome P450 2C43: cDNA cloning, heterologous expression, purification and characterization.
The cDNA of cytochrome P450 (CYP) 2C43 was cloned from cynomolgus monkey liver by RT-PCR. The deduced amino acid sequence showed 93% and 91% identity to human CYP2C9 and CYP2C19, respectively. The cDNA was expressed in Escherichia coli and purified by a series of chromatography steps, yielding a specific content of 11.5 nmol P450/mg protein. The substrate specificity of the purified CYP2C43 was examined in a reconstitution system comprising NADPH-P450 reductase, lipid, cytochrome b(5) and CYP2C marker substrates. The purified CYP2C43 showed high activity for testosterone 17-oxidation and progesterone 21-hydroxylation, which were also observed for CYP2C19 but not CYP2C9. In addition, CYP2C43 showed activity for (S)-mephenytoin 4'-hydroxylation, a marker reaction for CYP2C19. With CYP2C9 marker substrates, CYP2C43 exhibited low activity for diclofenac 4'-hydroxylation and no activity for tolbutamide p-methylhydroxylation. Therefore, in terms of substrate specificity, our results indicate that CYP2C43 is similar to CYP2C19, rather than CYP2C9.
Mitsuda
Maori
M
Department of Biology, Graduate School of Science, Osaka University, 2-17-85 Jusohonmachi, Yodogawa-ku, Osaka 532-8686.
Iwasaki
Masahiko
M
Asahi
Satoru
S
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D006224:Cricetinae; D065731:Cytochrome P-450 CYP2C19; D065729:Cytochrome P-450 CYP2C9; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D004008:Diclofenac / Q000493:pharmacokinetics; D006168:Guinea Pigs; D006801:Humans; D007700:Kinetics; D008252:Macaca fascicularis; D008617:Mephenytoin / Q000493:pharmacokinetics; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000302:isolation & purification / Q000378:metabolism; D011374:Progesterone / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D013739:Testosterone / Q000378:metabolism; D014044:Tolbutamide / Q000493:pharmacokinetics
D011994:Recombinant Proteins; C400322:cytochrome P-450 CYP2C subfamily; D004008:Diclofenac; D013739:Testosterone; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; D014044:Tolbutamide; D006899:Mixed Function Oxygenases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19; D008617:Mephenytoin