Heterologous Expression of Cytochrome P450 2D6 Mutants, Electron Transfer, and Catalysis of Bufuralol Hydroxylation: The Role of Aspartate 301 in Structural Integrity

doi:10.1006/abbi.2001.2510

Archives of Biochemistry and Biophysics

Volume 393, Issue 2, 15 September 2001, Pages 255-261

https://doi.org/10.1006/abbi.2001.2510 Get rights and content

Abstract

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.

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Aspirin (acetylsalicylic acid) is a well-known and widely-used analgesic. It is rapidly deacetylated to salicylic acid, which forms two hippuric acids—salicyluric acid and gentisuric acid—and two glucuronides. The oxidation of aspirin and salicylic acid has been reported with human liver microsomes, but data on individual cytochromes P450 involved in oxidation is lacking. In this study we monitored oxidation of these compounds by human liver microsomes and cytochrome P450 (P450) using UPLC with fluorescence detection. Microsomal oxidation of salicylic acid was much faster than aspirin. The two oxidation products were 2,5-dihydroxybenzoic acid (gentisic acid, documented by its UV and mass spectrum) and 2,3-dihydroxybenzoic acid. Formation of neither product was inhibited by desferrioxamine, suggesting a lack of contribution of oxygen radicals under these conditions. Although more liphophilic, aspirin was oxidized less efficiently, primarily to the 2,5-dihydroxy product. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5-hydroxylation of salicylic acid. Inhibitor studies with human liver microsomes indicated that all six of the previously mentioned P450s could contribute to both the 5- and 3-hydroxylation of salicylic acid and that P450s 2A6 and 2B6 have contributions to 5-hydroxylation. Inhibitor studies indicated that the major human P450 involved in both 3- and 5-hydroxylation of salicylic acid is P450 2E1.
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Results: Mitochondrial CYP2D6 supported by adrenodoxin/adrenodoxin reductase efficiently catalyzed MPTP to MPP⁺.
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^☆: This study was supported in part by the United States Public Health Service (USPHS) Grants R35 CA44353, R01 CA90426, and P30 ES00267. Nucleotide sequence analysis was supported in part by the Vanderbilt Cancer Center and USPHS Grant P30 CA68485.

²: Recipient of USPHS Postdoctoral Fellowship F32 CA79162. Present address: Department of Drug Metabolism and Safety Assessment, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033.

³: Present address: Department of Drug Metabolism, Merck & Company, 126 East Lincoln Avenue, Rahway, NJ 07065.

⁴: To whom correspondence and reprint requests should be addressed at Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Building, 23rd and Pierce Avenues, Nashville, TN 37232-0146. Fax: (615) 322-3141. E-mail: [email protected].

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Regular ArticleHeterologous Expression of Cytochrome P450 2D6 Mutants, Electron Transfer, and Catalysis of Bufuralol Hydroxylation: The Role of Aspartate 301 in Structural Integrity☆

Abstract

J. Biol. Chem.

Chem.-Biol. Interact.

Lancet

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Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

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Anal. Biochem.

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Arch. Biochem. Biophys.

J. Biol. Chem.

Pharmacogenetics

Drug Metab. Rev.

Biochemistry

Drug Metab. Rev.

Clin. Pharmacol. Ther.

Regular Article
Heterologous Expression of Cytochrome P450 2D6 Mutants, Electron Transfer, and Catalysis of Bufuralol Hydroxylation: The Role of Aspartate 301 in Structural Integrity☆