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 |
Externally published | Yes |
Keywords
- CYP2C19
- Enantiomer
- Glutamic acid-300
- Hexobarbital
- Phenylalanine-476
- Site-directed mutagenesis
- Substrate enantioselectivity
ASJC Scopus subject areas
- Analytical Chemistry
- Catalysis
- Pharmacology
- Drug Discovery
- Spectroscopy
- Organic Chemistry