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

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1 Citation (Scopus)

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

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

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    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, 172(1), 11-21. https://doi.org/10.1016/j.cbi.2007.11.010