title: 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.
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.
authors.affiliation: Laboratory of Health Chemistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan. shizuo@pharm.okayama-u.ac.jp
authors.forename: Shizuo
authors.initials: S
authors.lastname: Narimatsu
authors.forename: Kimio
authors.initials: K
authors.lastname: Kiryu
authors.forename: Rei
authors.initials: R
authors.lastname: Yonemoto
authors.forename: Manabu
authors.initials: M
authors.lastname: Yoshino
authors.forename: Mitsuko
authors.initials: M
authors.lastname: Kobatake
authors.forename: Daichi
authors.initials: D
authors.lastname: Kazamori
authors.forename: Saori
authors.initials: S
authors.lastname: Hagino
authors.forename: Kazufumi
authors.initials: K
authors.lastname: Masuda
authors.forename: Takashi
authors.initials: T
authors.lastname: Katsu
authors.forename: Masato
authors.initials: M
authors.lastname: Asanuma
authors.forename: Takuya
authors.initials: T
authors.lastname: Kumamoto
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Ishikawa
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Funae
authors.forename: Shigeru
authors.initials: S
authors.lastname: Yamano
authors.forename: Nobumitsu
authors.initials: N
authors.lastname: Hanioka
authors.forename: Shinsaku
authors.initials: S
authors.lastname: Naito
mesh_terms: D008735:5-Methoxytryptamine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D000072467:Cytochrome P450 Family 2; D004795:Enzyme Stability; D005786:Gene Expression Regulation; D008861:Microsomes; D015394:Molecular Structure; D009154:Mutation; D010084:Oxidation-Reduction; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
chemical_list: C033696:5-methoxy-N,N-diisopropyltryptamine; D008735:5-Methoxytryptamine; D000429:Alcohol Oxidoreductases; D001189:Aryl Hydrocarbon Hydroxylases; C504568:Cyp2d1 protein, rat; C119985:Cyp2d2 protein, rat; D000072467:Cytochrome P450 Family 2