Heterologous expression of cytochrome P450 2D6 mutants, electron transfer, and catalysis of bufuralol hydroxylation: the role of aspartate 301 in structural integrity.
Cytochrome P450 (P450) 2D6 is a polymorphic human enzyme involved in the oxidation of >50 drugs, most of which contain a basic nitrogen. In confirmation of previous work by others, substitutions at Asp301 decreased rates of substrate oxidation by P450 2D6. An anionic residue (Asp, Glu) at this position was found to be important in proper protein folding and heme incorporation, and positively charged residues were particularly disruptive in bacterial and also in baculovirus expression systems. Truncation of 20 N-terminal amino acids had no significant effect on catalytic activity except to attenuate P450 2D6 interaction with membranes and NADPH-P450 reductase. The truncation of the N-terminus increased the level of bacterial expression of wild-type P450 2D6 (Asp301) but markedly reduced expression of all codon 301 mutants, including Glu301. Reduction of ferric P450 2D6 by NADPH-P450 reductase was enhanced in the presence of the prototypic substrate bufuralol. Bacterial flavodoxin, an NADPH-P450 reductase homolog, binds tightly to P450 2D6 but is inefficient in electron transfer to the heme. These results collectively indicate that the acidic residue at position 301 in P450 2D6 has a structural role in addition to any in substrate binding and that the N-terminus of P450 2D6 is relatively unimportant to catalytic activity beyond a role in facilitating binding to NADPH-P450 reductase.
Hanna
I H
IH
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Kim
M S
MS
Guengerich
F P
FP
D000319:Adrenergic beta-Antagonists / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D002384:Catalysis; D019389:Cytochrome P-450 CYP2D6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004579:Electron Transport; D004926:Escherichia coli; D004983:Ethanolamines / Q000378:metabolism*; D005290:Ferric Compounds / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression; D006900:Hydroxylation; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation
D000319:Adrenergic beta-Antagonists; D018076:DNA, Complementary; D004983:Ethanolamines; D005290:Ferric Compounds; D001224:Aspartic Acid; C010831:bufuralol; D019389:Cytochrome P-450 CYP2D6