Functional characterization of single nucleotide polymorphisms with amino acid substitution in CYP1A2, CYP2A6, and CYP2B6 found in the Japanese population.
As a part of the studies conducted by the Pharma SNPs Consortium (PSC), the enzyme activities of CYP1A2, CYP2A6 and CYP2B6 variants with altered amino acids as a result of single nucleotide polymorphisms (SNPs) found among the Japanese population were analyzed under a unified protocol using the same lots of reagents by the laboratories participating in the PSC. Mutations in CYP1A2, CYP2A6 and CYP2B6 were introduced by site-directed mutagenesis and the wild type and mutated CYP molecules were expressed in Escherichia coli. The expressed cytochrome P450s were purified and the enzyme activities were measured in reconstitution systems. CYP1A2 and CYP1A2Gln478His did not show any differences in 7-ethoxyresorufin O-deethylase activity. CYP2A6 and CYP2A6Glu419Asp metabolized coumarin to form 7-hydroxycoumarin in a similar manner, whereas CYP2A6Ile471Thr showed low activity compared to the wild-type CYP2A6. CYP2B6, CYP2B6Pro167Ala and CYP2B6Arg487Cys showed the same activity for 7-ethoxy-4-triflouromethyl-coumarin O-deethylation. However, CYP2B6Gln172His was roughly twice as active as CYP2B6 and the other CYP2B6 variants for 7-ethoxy-4-triflouromethylcoumarin O-deethylation activity. Although higher inter- and intra-laboratory variations were observed for the calculated Km and V(max) values because the studies were conducted in several different laboratories, the degree of variations was reduced by the increased number of analyses and the adoption of a simple analysis system.
Iwasaki
Masahiko
M
Graduate School of Osaka University, Japan. Iwasaki_Masahiko@takeda.co.jp
Yoshimura
Yoshinobu
Y
Asahi
Satoru
S
Saito
Kimitoshi
K
Sakai
Shuichi
S
Morita
Shigemichi
S
Takenaka
Osamu
O
Inoda
Toshio
T
Kashiyama
Eiji
E
Aoyama
Akinori
A
Nakabayashi
Takeshi
T
Omori
Satoshi
S
Kuwabara
Takashi
T
Izumi
Takashi
T
Nakamura
Kouichi
K
Takanaka
Kaoru
K
Nakayama
Yukiharu
Y
Takeuchi
Mitsuaki
M
Nakamura
Hideki
H
Kametani
Shunichi
S
Terauchi
Yoshiaki
Y
Hashizume
Takanori
T
Nagayama
Sekio
S
Kume
Toshiyuki
T
Achira
Meguru
M
Kawai
Hiroyuki
H
Kawashiro
Takashi
T
Nakamura
Akio
A
Nakai
Yasuhiro
Y
Kagayama
Akira
A
Shiraga
Toshifumi
T
Niwa
Takuro
T
Yoshimura
Takuya
T
Morita
Jun
J
Ohsawa
Fukuichi
F
Tani
Masato
M
Osawa
Nobuo
N
Ida
Keiichi
K
Noguchi
Kiyoshi
K
D019943:Amino Acid Substitution; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism*; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics* / Q000378:metabolism*; D065637:Cytochrome P-450 CYP2A6; D065702:Cytochrome P-450 CYP2B6; D004926:Escherichia coli / Q000201:enzymology; D005787:Gene Frequency; D006801:Humans; D007564:Japan; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; C585599:CYP2B6 protein, human; D019388:Cytochrome P-450 CYP1A2; D065637:Cytochrome P-450 CYP2A6; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating; D009251:NADPH-Ferrihemoprotein Reductase