title: Stereoselective hexobarbital 3'-hydroxylation by CYP2C19 expressed in yeast cells and the roles of amino acid residues at positions 300 and 476.
abstract: We examined the enzymatic function of recombinant CYP2C19 in enantiomeric hexobarbital (HB) 3'-hydroxylation, and searched the roles of amino acid residues, such as Phe-100, Phe-114, Asp-293, Glu-300, and Phe-476 of CYP2C19 in the stereoselective HB 3'-hydroxylation, using a yeast cell expression system and site-directed mutagenesis method. CYP2C19 wild-type exerted substrate enantioselectivity of (R)-HB>>(S)-HB and metabolite diastereoselectivity of 3'(R)<3'(S) in 3'-hydroxylation of HB enantiomers. The substitution of Asp-293 by alanine failed to yield an observable peak at 450 nm in its reduced carbon monoxide-difference spectrum. CYP2C19-E300A and CYP2C19-E300V with alanine and valine, respectively, in place of Glu-300 exerted total HB 3'-hydroxylation activities of 45 and 108%, respectively, that of the wild-type. Interestingly, these two mutants showed substrate enantioselectivity of (R)-HB<(S)-HB, which is opposite to that of the wild-type, while metabolite diasteroselectivity remained unchanged. The replacement of Phe-476 by alanine increased total HB 3'-hydroxylation activity to approximately 3-fold that of the wild-type. Particularly, 3'(S)-OH-(S)-HB-forming activity elevated to 7-fold that of the wild-type, resulting in the reversal of the substrate enantioselectivity. In contrast, the substitution of phenylalanine at positions 100 and 114 by alanine did not produce a remarkable change in the total activity or the substrate enantioselectivity. These results indicate that Glu-300 and Phe-476 are important in stereoselective oxidation of HB enantiomers by CYP2C19.
authors.affiliation: Laboratory of Health Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan.
authors.forename: Keita
authors.initials: K
authors.lastname: Saito
authors.forename: Hikari
authors.initials: H
authors.lastname: Dan
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Masuda
authors.forename: Takashi
authors.initials: T
authors.lastname: Katsu
authors.forename: Nobumitsu
authors.initials: N
authors.lastname: Hanioka
authors.forename: Shigeo
authors.initials: S
authors.lastname: Yamamoto
authors.forename: Kazuko
authors.initials: K
authors.lastname: Miyano
authors.forename: Shigeru
authors.initials: S
authors.lastname: Yamano
authors.forename: Shizuo
authors.initials: S
authors.lastname: Narimatsu
mesh_terms: D019943:Amino Acid Substitution; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D065731:Cytochrome P-450 CYP2C19; D017931:DNA Primers / Q000235:genetics; D006591:Hexobarbital / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D006591:Hexobarbital; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19