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Journal of Biochemistry Advance Access originally published online on May 28, 2008
Journal of Biochemistry 2008 144(3):323-333; doi:10.1093/jb/mvn070
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© 2008 The Japanese Biochemical Society

Important Amino Acid Residues that Confer CYP2C19 Selective Activity to CYP2C9

Yasunobu Wada1, Maori Mitsuda1, Yasuhiro Ishihara2, Masatomo Watanabe3, Masahiko Iwasaki1,* and Satoru Asahi4

1Department of Biology, Graduate School of Science, Osaka University, Osaka; 2Laboratory of Pharmacology; 3Laboratory of Molecular Pharmacology, Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Sanuki; and 4Development Research Center, Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, Osaka, Japan

*To whom correspondence should be addressed. Tel: +81-(6)-6300-6697, Fax: +81-(6)-6300-6918, E-mail: iwasaki_masahiko{at}takeda.co.jp

Received April 18, 2008; Accepted May 19, 2008


  Abstract

Although CYP2C9 and CYP2C19 display 91% sequence identity at the amino acid level, the two enzymes have distinct substrate specificities for compounds such as diclofenac, progesterone and (S)-mephenytoin. Amino acid substitutions in CYP2C9 were made based on an alignment of CYP2C9, CYP2C19 and monkey CYP2C43 sequences. Mutants of CYP2C9 were expressed in Escherichia coli. Sixteen amino acids, which are common to both CYP2C19 and CYP2C43 but different between CYP2C9 and CYP2C19, were substituted in CYP2C9 (CYP2C9-16aa). Next, the mutated amino acids in CYP2C9-16aa were individually reverted to those of CYP2C9 to examine the effect of each substitution on the enzymatic activity for CYP2C marker substrates. In addition, the role of the F–G loop in CYP2C9 and CYP2C19 was examined for substrate specificity and enzymatic activity. Our results showed: (i) CYP2C9-16aa displays 11% (S)-mephenytoin 4'-hydroxylase and full omeprazole 5-hydroxylase activity compared with that of CYP2C19; (ii) residue 286 is important for conferring CYP2C9-like enzyme activity on CYP2C9-16aa and residue 442 in CYP2C19 may be involved in the interaction with NADPH-P450 reductase; (iii) substitution of the F–G loop in CYP2C9 to that of CYP2C19 enhances tolbutamide p-methyhydroxylase and diclofenac 4'-hydroxylase activities and confers partial (S)-mephenytoin 4'-hydroxylase and omeprazole 5-hydroxylase activities, which are attributed to CYP2C19.

Key Words: CYP2C9, CYP2C19, CYP2C43, F–G loop, NADPH-P450 reductase, substrate specificity

Abbreviations: ABSF, 4-(2-aminoethyl)benzenesulfonyl fluoride·HCl; CYP, cytochrome P450; DTT, dithiothreitol; IPTG, isopropyl-β-D-thiogalactopyranoside; NADPH, nicotinamide adenine dinucleotide phosphate, reduced form; PCR, polymerase chain reaction


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