The roles of amino acid residues at positions 216 and 219 in the structural stability and metabolic functions of rat cytochrome P450 2D1 and 2D2

Shizuo Narimatsu, Kimio Kiryu, Rei Yonemoto, Manabu Yoshino, Mitsuko Kobatake, Daichi Kazamori, Saori Hagino, Kazufumi Masuda, Takashi Katsu, Masato Asanuma, Takuya Kumamoto, Tsutomu Ishikawa, Yoshihiko Funae, Shigeru Yamano, Nobumitsu Hanioka, Shinsaku Naito

Research output: Contribution to journalArticle

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Abstract

We examined the effects of the mutual substitution of amino acid residues at positions 216 and 219 between rat CYP2D1 and CYP2D2 on their microsomal contents and enzymatic functions using a yeast cell expression system and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) as a substrate. CYP2D1 has amino acid residues, leucine and valine, at positions of 216 and 219, respectively, whereas CYP2D2 has phenylalanine and aspartic acid at the same positions. In reduced carbon monoxide-difference spectroscopic analysis, the substitution of Asp-219 of CYP2D2 by valine markedly increased a peak at 450 nm and concomitantly decreased a peak at 420 nm, while the replacement of Phe-216 of CYP2D2 with leucine gave no observable change. The double substitution of Phe-216 and Asp-219 by leucine and valine, respectively, yielded a typical CYP spectrum. The substitution of Val-219 of CYP2D1 by aspartic acid decreased the CYP content to one-half, whereas the replacement of Leu-216 with phenylalanine did not have any effect. The double substitution of Leu-216 and Val-219 of CYP2D1 by phenylalanine and aspartic acid, respectively, diminished the CYP content by 90%. CYP2D1 catalyzed both 5-MeO-DIPT N-deisopropylation and O-demethylation at relatively low levels, while CYP2D2 catalyzed 5-MeO-DIPT O-demethylation efficiently. The substitution of the amino acid at position 216 substantially increased 5-MeO-DIPT oxidation activities of the two CYP2D enzymes. The replacement of the amino acid at position 219 increased the 5-MeO-DIPT O- and N-dealkylation activities of CYP2D1, whereas it decreased the 5-MeO-DIPT O-demethylation activity of CYP2D2. These results indicate that amino acid residues at positions 216 and 219 have important roles in the enzymatic functions of rat CYP2D1 and CYP2D2.

Original languageEnglish
Pages (from-to)11-21
Number of pages11
JournalChemico-Biological Interactions
Volume172
Issue number1
DOIs
Publication statusPublished - Mar 10 2008

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Cytochrome P-450 Enzyme System
Rats
Substitution reactions
Amino Acids
Valine
Phenylalanine
Aspartic Acid
Leucine
Amino Acid Substitution
Dealkylation
Spectroscopic analysis
Carbon Monoxide
Yeast
5-methoxy-N,N-diisopropyltryptamine
Yeasts
Cells
Oxidation
Substrates
Enzymes

Keywords

  • 5-Methoxy-N,N-diisopropyltryptamine
  • Amino acids at positions 216 and 219
  • CYP2D1
  • CYP2D2
  • Protein stability
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Toxicology

Cite this

The roles of amino acid residues at positions 216 and 219 in the structural stability and metabolic functions of rat cytochrome P450 2D1 and 2D2. / Narimatsu, Shizuo; Kiryu, Kimio; Yonemoto, Rei; Yoshino, Manabu; Kobatake, Mitsuko; Kazamori, Daichi; Hagino, Saori; Masuda, Kazufumi; Katsu, Takashi; Asanuma, Masato; Kumamoto, Takuya; Ishikawa, Tsutomu; Funae, Yoshihiko; Yamano, Shigeru; Hanioka, Nobumitsu; Naito, Shinsaku.

In: Chemico-Biological Interactions, Vol. 172, No. 1, 10.03.2008, p. 11-21.

Research output: Contribution to journalArticle

Narimatsu, S, Kiryu, K, Yonemoto, R, Yoshino, M, Kobatake, M, Kazamori, D, Hagino, S, Masuda, K, Katsu, T, Asanuma, M, Kumamoto, T, Ishikawa, T, Funae, Y, Yamano, S, Hanioka, N & Naito, S 2008, 'The roles of amino acid residues at positions 216 and 219 in the structural stability and metabolic functions of rat cytochrome P450 2D1 and 2D2', Chemico-Biological Interactions, vol. 172, no. 1, pp. 11-21. https://doi.org/10.1016/j.cbi.2007.11.010
Narimatsu, Shizuo ; Kiryu, Kimio ; Yonemoto, Rei ; Yoshino, Manabu ; Kobatake, Mitsuko ; Kazamori, Daichi ; Hagino, Saori ; Masuda, Kazufumi ; Katsu, Takashi ; Asanuma, Masato ; Kumamoto, Takuya ; Ishikawa, Tsutomu ; Funae, Yoshihiko ; Yamano, Shigeru ; Hanioka, Nobumitsu ; Naito, Shinsaku. / The roles of amino acid residues at positions 216 and 219 in the structural stability and metabolic functions of rat cytochrome P450 2D1 and 2D2. In: Chemico-Biological Interactions. 2008 ; Vol. 172, No. 1. pp. 11-21.
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T1 - The roles of amino acid residues at positions 216 and 219 in the structural stability and metabolic functions of rat cytochrome P450 2D1 and 2D2

AU - Narimatsu, Shizuo

AU - Kiryu, Kimio

AU - Yonemoto, Rei

AU - Yoshino, Manabu

AU - Kobatake, Mitsuko

AU - Kazamori, Daichi

AU - Hagino, Saori

AU - Masuda, Kazufumi

AU - Katsu, Takashi

AU - Asanuma, Masato

AU - Kumamoto, Takuya

AU - Ishikawa, Tsutomu

AU - Funae, Yoshihiko

AU - Yamano, Shigeru

AU - Hanioka, Nobumitsu

AU - Naito, Shinsaku

PY - 2008/3/10

Y1 - 2008/3/10

N2 - We examined the effects of the mutual substitution of amino acid residues at positions 216 and 219 between rat CYP2D1 and CYP2D2 on their microsomal contents and enzymatic functions using a yeast cell expression system and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) as a substrate. CYP2D1 has amino acid residues, leucine and valine, at positions of 216 and 219, respectively, whereas CYP2D2 has phenylalanine and aspartic acid at the same positions. In reduced carbon monoxide-difference spectroscopic analysis, the substitution of Asp-219 of CYP2D2 by valine markedly increased a peak at 450 nm and concomitantly decreased a peak at 420 nm, while the replacement of Phe-216 of CYP2D2 with leucine gave no observable change. The double substitution of Phe-216 and Asp-219 by leucine and valine, respectively, yielded a typical CYP spectrum. The substitution of Val-219 of CYP2D1 by aspartic acid decreased the CYP content to one-half, whereas the replacement of Leu-216 with phenylalanine did not have any effect. The double substitution of Leu-216 and Val-219 of CYP2D1 by phenylalanine and aspartic acid, respectively, diminished the CYP content by 90%. CYP2D1 catalyzed both 5-MeO-DIPT N-deisopropylation and O-demethylation at relatively low levels, while CYP2D2 catalyzed 5-MeO-DIPT O-demethylation efficiently. The substitution of the amino acid at position 216 substantially increased 5-MeO-DIPT oxidation activities of the two CYP2D enzymes. The replacement of the amino acid at position 219 increased the 5-MeO-DIPT O- and N-dealkylation activities of CYP2D1, whereas it decreased the 5-MeO-DIPT O-demethylation activity of CYP2D2. These results indicate that amino acid residues at positions 216 and 219 have important roles in the enzymatic functions of rat CYP2D1 and CYP2D2.

AB - We examined the effects of the mutual substitution of amino acid residues at positions 216 and 219 between rat CYP2D1 and CYP2D2 on their microsomal contents and enzymatic functions using a yeast cell expression system and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) as a substrate. CYP2D1 has amino acid residues, leucine and valine, at positions of 216 and 219, respectively, whereas CYP2D2 has phenylalanine and aspartic acid at the same positions. In reduced carbon monoxide-difference spectroscopic analysis, the substitution of Asp-219 of CYP2D2 by valine markedly increased a peak at 450 nm and concomitantly decreased a peak at 420 nm, while the replacement of Phe-216 of CYP2D2 with leucine gave no observable change. The double substitution of Phe-216 and Asp-219 by leucine and valine, respectively, yielded a typical CYP spectrum. The substitution of Val-219 of CYP2D1 by aspartic acid decreased the CYP content to one-half, whereas the replacement of Leu-216 with phenylalanine did not have any effect. The double substitution of Leu-216 and Val-219 of CYP2D1 by phenylalanine and aspartic acid, respectively, diminished the CYP content by 90%. CYP2D1 catalyzed both 5-MeO-DIPT N-deisopropylation and O-demethylation at relatively low levels, while CYP2D2 catalyzed 5-MeO-DIPT O-demethylation efficiently. The substitution of the amino acid at position 216 substantially increased 5-MeO-DIPT oxidation activities of the two CYP2D enzymes. The replacement of the amino acid at position 219 increased the 5-MeO-DIPT O- and N-dealkylation activities of CYP2D1, whereas it decreased the 5-MeO-DIPT O-demethylation activity of CYP2D2. These results indicate that amino acid residues at positions 216 and 219 have important roles in the enzymatic functions of rat CYP2D1 and CYP2D2.

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KW - Amino acids at positions 216 and 219

KW - CYP2D1

KW - CYP2D2

KW - Protein stability

KW - Site-directed mutagenesis

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