Change in enantioselectivity in bufuralol 1″-hydroxylation by the substitution of phenylalanine-120 by alanine in cytochrome P450 2D6

Kazufumi Masuda, Keietsu Tamagake, Yukie Okuda, Fumihiro Torigoe, Daisuke Tsuzuki, Takashi Isobe, Hiroyuki Hichiya, Nobumitsu Hanioka, Shigeo Yamamoto, Shizuo Narimatsu

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

The functional roles of phenylalanine at position 120 in drug oxidation by cytochrome P450 2D6 (CYP2D6) were examined using a yeast cell expression system and bufuralol (BF) enantiomers as a chiral substrate. Two mutated cDNAs, one encoding a CYP2D6 mutant having alanine instead of Phe-120 (F120A) and another encoding a mutant having alanine instead of Glu-222 (E222A), were prepared by site-directed mutagenesis and transformed into yeast cells via pGYRI vectors. The enantiomeric BF 1″-hydroxylase activities of the mutants were compared with those of the wild type. When enantiomeric BF 1″-hydroxylase activities at a substrate concentration of 100 μM were compared, the CYP2D6 wild type showed substrate enantioselectivity of (R-BF ≫ S-BF) and the F120A mutant exhibited substrate enantioselectivity of (R-BF ≤ S-BF), whereas the product diastereoselectivity of (1″-S-OH-BF ≪ 1″-S-OH-BF) was similar between the wild type and the mutant. The activities of the other mutant (E222A) were much lower than those of the wild type and the F120A mutant, while its substrate enantioselectivity and product diastereoselectivity were the same as those of the wild type. The kinetics demonstrated that apparent K m values were similar among the recombinant enzymes, and V max values clearly reflected the selectivity described above. These results indicate that Phe-120 has a key role in the enantioselective BF 1″-hydroxylation by CYP2D6.

Original languageEnglish
Pages (from-to)37-43
Number of pages7
JournalChirality
Volume17
Issue number1
DOIs
Publication statusPublished - 2005

Fingerprint

Cytochrome P-450 CYP2D6
Hydroxylation
Enantioselectivity
Phenylalanine
Alanine
Substitution reactions
Substrates
Yeast
Cells
Mutagenesis
Enantiomers
Yeasts
Enzymes
bufuralol
Cytochrome P-450 Enzyme System
Site-Directed Mutagenesis
Oxidation
Kinetics
Complementary DNA

Keywords

  • Bufuralol enantiomer
  • CYP2D6
  • Glutamic acid-222
  • Phenylalanine-120
  • Substrate enantioselectivity

ASJC Scopus subject areas

  • Analytical Chemistry
  • Drug Discovery
  • Organic Chemistry
  • Pharmacology

Cite this

Change in enantioselectivity in bufuralol 1″-hydroxylation by the substitution of phenylalanine-120 by alanine in cytochrome P450 2D6. / Masuda, Kazufumi; Tamagake, Keietsu; Okuda, Yukie; Torigoe, Fumihiro; Tsuzuki, Daisuke; Isobe, Takashi; Hichiya, Hiroyuki; Hanioka, Nobumitsu; Yamamoto, Shigeo; Narimatsu, Shizuo.

In: Chirality, Vol. 17, No. 1, 2005, p. 37-43.

Research output: Contribution to journalArticle

Masuda, K, Tamagake, K, Okuda, Y, Torigoe, F, Tsuzuki, D, Isobe, T, Hichiya, H, Hanioka, N, Yamamoto, S & Narimatsu, S 2005, 'Change in enantioselectivity in bufuralol 1″-hydroxylation by the substitution of phenylalanine-120 by alanine in cytochrome P450 2D6', Chirality, vol. 17, no. 1, pp. 37-43. https://doi.org/10.1002/chir.20092
Masuda, Kazufumi ; Tamagake, Keietsu ; Okuda, Yukie ; Torigoe, Fumihiro ; Tsuzuki, Daisuke ; Isobe, Takashi ; Hichiya, Hiroyuki ; Hanioka, Nobumitsu ; Yamamoto, Shigeo ; Narimatsu, Shizuo. / Change in enantioselectivity in bufuralol 1″-hydroxylation by the substitution of phenylalanine-120 by alanine in cytochrome P450 2D6. In: Chirality. 2005 ; Vol. 17, No. 1. pp. 37-43.
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abstract = "The functional roles of phenylalanine at position 120 in drug oxidation by cytochrome P450 2D6 (CYP2D6) were examined using a yeast cell expression system and bufuralol (BF) enantiomers as a chiral substrate. Two mutated cDNAs, one encoding a CYP2D6 mutant having alanine instead of Phe-120 (F120A) and another encoding a mutant having alanine instead of Glu-222 (E222A), were prepared by site-directed mutagenesis and transformed into yeast cells via pGYRI vectors. The enantiomeric BF 1″-hydroxylase activities of the mutants were compared with those of the wild type. When enantiomeric BF 1″-hydroxylase activities at a substrate concentration of 100 μM were compared, the CYP2D6 wild type showed substrate enantioselectivity of (R-BF ≫ S-BF) and the F120A mutant exhibited substrate enantioselectivity of (R-BF ≤ S-BF), whereas the product diastereoselectivity of (1″-S-OH-BF ≪ 1″-S-OH-BF) was similar between the wild type and the mutant. The activities of the other mutant (E222A) were much lower than those of the wild type and the F120A mutant, while its substrate enantioselectivity and product diastereoselectivity were the same as those of the wild type. The kinetics demonstrated that apparent K m values were similar among the recombinant enzymes, and V max values clearly reflected the selectivity described above. These results indicate that Phe-120 has a key role in the enantioselective BF 1″-hydroxylation by CYP2D6.",
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AU - Torigoe, Fumihiro

AU - Tsuzuki, Daisuke

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AB - The functional roles of phenylalanine at position 120 in drug oxidation by cytochrome P450 2D6 (CYP2D6) were examined using a yeast cell expression system and bufuralol (BF) enantiomers as a chiral substrate. Two mutated cDNAs, one encoding a CYP2D6 mutant having alanine instead of Phe-120 (F120A) and another encoding a mutant having alanine instead of Glu-222 (E222A), were prepared by site-directed mutagenesis and transformed into yeast cells via pGYRI vectors. The enantiomeric BF 1″-hydroxylase activities of the mutants were compared with those of the wild type. When enantiomeric BF 1″-hydroxylase activities at a substrate concentration of 100 μM were compared, the CYP2D6 wild type showed substrate enantioselectivity of (R-BF ≫ S-BF) and the F120A mutant exhibited substrate enantioselectivity of (R-BF ≤ S-BF), whereas the product diastereoselectivity of (1″-S-OH-BF ≪ 1″-S-OH-BF) was similar between the wild type and the mutant. The activities of the other mutant (E222A) were much lower than those of the wild type and the F120A mutant, while its substrate enantioselectivity and product diastereoselectivity were the same as those of the wild type. The kinetics demonstrated that apparent K m values were similar among the recombinant enzymes, and V max values clearly reflected the selectivity described above. These results indicate that Phe-120 has a key role in the enantioselective BF 1″-hydroxylation by CYP2D6.

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