Stereoselective hexobarbital 3′-hydroxylation by CYP2C19 expressed in yeast cells and the roles of amino acid residues at positions 300 and 476

Keita Saito, Hikari Dan, Kazufumi Masuda, Takashi Katsu, Nobumitsu Hanioka, Shigeo Yamamoto, Kazuko Miyano, Shigeru Yamano, Shizuo Narimatsu

Research output: Contribution to journalArticle

3 Citations (Scopus)

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 languageEnglish
Pages (from-to)550-558
Number of pages9
JournalChirality
Volume19
Issue number7
DOIs
Publication statusPublished - 2007

Fingerprint

Hexobarbital
Hydroxylation
Yeast
Enantioselectivity
Amino acids
Yeasts
Cells
Amino Acids
Enantiomers
Substrates
Metabolites
Alanine
Substitution reactions
Mutagenesis
Carbon monoxide
Cytochrome P-450 CYP2C19
Oxidation
Valine
Carbon Monoxide
Site-Directed Mutagenesis

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 journalArticle

Saito, K, Dan, H, Masuda, K, Katsu, T, Hanioka, N, Yamamoto, S, Miyano, K, Yamano, S & Narimatsu, S 2007, 'Stereoselective hexobarbital 3′-hydroxylation by CYP2C19 expressed in yeast cells and the roles of amino acid residues at positions 300 and 476', Chirality, vol. 19, no. 7, pp. 550-558. https://doi.org/10.1002/chir.20412
Saito, Keita ; Dan, Hikari ; Masuda, Kazufumi ; Katsu, Takashi ; Hanioka, Nobumitsu ; Yamamoto, Shigeo ; Miyano, Kazuko ; Yamano, Shigeru ; Narimatsu, Shizuo. / Stereoselective hexobarbital 3′-hydroxylation by CYP2C19 expressed in yeast cells and the roles of amino acid residues at positions 300 and 476. In: Chirality. 2007 ; Vol. 19, No. 7. pp. 550-558.
@article{a8dc01a6c3b6407b83ec2a495eefd6d2,
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.",
keywords = "CYP2C19, Enantiomer, Glutamic acid-300, Hexobarbital, Phenylalanine-476, Site-directed mutagenesis, Substrate enantioselectivity",
author = "Keita Saito and Hikari Dan and Kazufumi Masuda and Takashi Katsu and Nobumitsu Hanioka and Shigeo Yamamoto and Kazuko Miyano and Shigeru Yamano and Shizuo Narimatsu",
year = "2007",
doi = "10.1002/chir.20412",
language = "English",
volume = "19",
pages = "550--558",
journal = "Chirality",
issn = "0899-0042",
publisher = "Wiley-Liss Inc.",
number = "7",

}

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 -