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.
| Original language | English |
|---|---|
| Pages (from-to) | 550-558 |
| Number of pages | 9 |
| Journal | Chirality |
| Volume | 19 |
| Issue number | 7 |
| DOIs | |
| Publication status | Published - 2007 |
Fingerprint
Keywords
- CYP2C19
- Enantiomer
- Glutamic acid-300
- Hexobarbital
- Phenylalanine-476
- Site-directed mutagenesis
- Substrate enantioselectivity
ASJC Scopus subject areas
- Analytical Chemistry
- Drug Discovery
- Organic Chemistry
- Pharmacology
Cite this
Stereoselective hexobarbital 3′-hydroxylation by CYP2C19 expressed in yeast cells and the roles of amino acid residues at positions 300 and 476. / Saito, Keita; Dan, Hikari; Masuda, Kazufumi; Katsu, Takashi; Hanioka, Nobumitsu; Yamamoto, Shigeo; Miyano, Kazuko; Yamano, Shigeru; Narimatsu, Shizuo.
In: Chirality, Vol. 19, No. 7, 2007, p. 550-558.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Stereoselective hexobarbital 3′-hydroxylation by CYP2C19 expressed in yeast cells and the roles of amino acid residues at positions 300 and 476
AU - Saito, Keita
AU - Dan, Hikari
AU - Masuda, Kazufumi
AU - Katsu, Takashi
AU - Hanioka, Nobumitsu
AU - Yamamoto, Shigeo
AU - Miyano, Kazuko
AU - Yamano, Shigeru
AU - Narimatsu, Shizuo
PY - 2007
Y1 - 2007
N2 - 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.
AB - 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.
KW - CYP2C19
KW - Enantiomer
KW - Glutamic acid-300
KW - Hexobarbital
KW - Phenylalanine-476
KW - Site-directed mutagenesis
KW - Substrate enantioselectivity
UR - http://www.scopus.com/inward/record.url?scp=34250831484&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34250831484&partnerID=8YFLogxK
U2 - 10.1002/chir.20412
DO - 10.1002/chir.20412
M3 - Article
C2 - 17487889
AN - SCOPUS:34250831484
VL - 19
SP - 550
EP - 558
JO - Chirality
JF - Chirality
SN - 0899-0042
IS - 7
ER -