title: Functional characterization of single nucleotide polymorphisms with amino acid substitution in CYP1A2, CYP2A6, and CYP2B6 found in the Japanese population.
abstract: 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.
authors.affiliation: Graduate School of Osaka University, Japan. Iwasaki_Masahiko@takeda.co.jp
authors.forename: Masahiko
authors.initials: M
authors.lastname: Iwasaki
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Yoshimura
authors.forename: Satoru
authors.initials: S
authors.lastname: Asahi
authors.forename: Kimitoshi
authors.initials: K
authors.lastname: Saito
authors.forename: Shuichi
authors.initials: S
authors.lastname: Sakai
authors.forename: Shigemichi
authors.initials: S
authors.lastname: Morita
authors.forename: Osamu
authors.initials: O
authors.lastname: Takenaka
authors.forename: Toshio
authors.initials: T
authors.lastname: Inoda
authors.forename: Eiji
authors.initials: E
authors.lastname: Kashiyama
authors.forename: Akinori
authors.initials: A
authors.lastname: Aoyama
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nakabayashi
authors.forename: Satoshi
authors.initials: S
authors.lastname: Omori
authors.forename: Takashi
authors.initials: T
authors.lastname: Kuwabara
authors.forename: Takashi
authors.initials: T
authors.lastname: Izumi
authors.forename: Kouichi
authors.initials: K
authors.lastname: Nakamura
authors.forename: Kaoru
authors.initials: K
authors.lastname: Takanaka
authors.forename: Yukiharu
authors.initials: Y
authors.lastname: Nakayama
authors.forename: Mitsuaki
authors.initials: M
authors.lastname: Takeuchi
authors.forename: Hideki
authors.initials: H
authors.lastname: Nakamura
authors.forename: Shunichi
authors.initials: S
authors.lastname: Kametani
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Terauchi
authors.forename: Takanori
authors.initials: T
authors.lastname: Hashizume
authors.forename: Sekio
authors.initials: S
authors.lastname: Nagayama
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Kume
authors.forename: Meguru
authors.initials: M
authors.lastname: Achira
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kawai
authors.forename: Takashi
authors.initials: T
authors.lastname: Kawashiro
authors.forename: Akio
authors.initials: A
authors.lastname: Nakamura
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Nakai
authors.forename: Akira
authors.initials: A
authors.lastname: Kagayama
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Shiraga
authors.forename: Takuro
authors.initials: T
authors.lastname: Niwa
authors.forename: Takuya
authors.initials: T
authors.lastname: Yoshimura
authors.forename: Jun
authors.initials: J
authors.lastname: Morita
authors.forename: Fukuichi
authors.initials: F
authors.lastname: Ohsawa
authors.forename: Masato
authors.initials: M
authors.lastname: Tani
authors.forename: Nobuo
authors.initials: N
authors.lastname: Osawa
authors.forename: Keiichi
authors.initials: K
authors.lastname: Ida
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Noguchi
mesh_terms: 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*
chemical_list: 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