Important amino acid residues that confer CYP2C19 selective activity to CYP2C9.
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.
Department of Biology, Graduate School of Science, Osaka University, Osaka, Japan.
Yasunobu
Y
Wada
Maori
M
Mitsuda
Yasuhiro
Y
Ishihara
Masatomo
M
Watanabe
Masahiko
M
Iwasaki
Satoru
S
Asahi
D000595:Amino Acid Sequence; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry*; D001671:Biochemistry / Q000379:methods; D065731:Cytochrome P-450 CYP2C19; D065729:Cytochrome P-450 CYP2C9; D017931:DNA Primers / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000737:chemistry; D013379:Substrate Specificity
D017931:DNA Primers; D009249:NADP; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19