Tags

Type your tag names separated by a space and hit enter

Heterologous expression of cytochrome P450 2D6 mutants, electron transfer, and catalysis of bufuralol hydroxylation: the role of aspartate 301 in structural integrity.
Arch Biochem Biophys. 2001 Sep 15; 393(2):255-61.AB

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.

Authors+Show Affiliations

Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

11556812

Citation

Hanna, I H., et al. "Heterologous Expression of Cytochrome P450 2D6 Mutants, Electron Transfer, and Catalysis of Bufuralol Hydroxylation: the Role of Aspartate 301 in Structural Integrity." Archives of Biochemistry and Biophysics, vol. 393, no. 2, 2001, pp. 255-61.
Hanna IH, Kim MS, Guengerich FP. Heterologous expression of cytochrome P450 2D6 mutants, electron transfer, and catalysis of bufuralol hydroxylation: the role of aspartate 301 in structural integrity. Arch Biochem Biophys. 2001;393(2):255-61.
Hanna, I. H., Kim, M. S., & Guengerich, F. P. (2001). Heterologous expression of cytochrome P450 2D6 mutants, electron transfer, and catalysis of bufuralol hydroxylation: the role of aspartate 301 in structural integrity. Archives of Biochemistry and Biophysics, 393(2), 255-61.
Hanna IH, Kim MS, Guengerich FP. Heterologous Expression of Cytochrome P450 2D6 Mutants, Electron Transfer, and Catalysis of Bufuralol Hydroxylation: the Role of Aspartate 301 in Structural Integrity. Arch Biochem Biophys. 2001 Sep 15;393(2):255-61. PubMed PMID: 11556812.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Heterologous expression of cytochrome P450 2D6 mutants, electron transfer, and catalysis of bufuralol hydroxylation: the role of aspartate 301 in structural integrity. AU - Hanna,I H, AU - Kim,M S, AU - Guengerich,F P, PY - 2001/9/15/pubmed PY - 2001/10/19/medline PY - 2001/9/15/entrez SP - 255 EP - 61 JF - Archives of biochemistry and biophysics JO - Arch Biochem Biophys VL - 393 IS - 2 N2 - 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. SN - 0003-9861 UR - https://www.unboundmedicine.com/medline/citation/11556812/Heterologous_expression_of_cytochrome_P450_2D6_mutants_electron_transfer_and_catalysis_of_bufuralol_hydroxylation:_the_role_of_aspartate_301_in_structural_integrity_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0003-9861(01)92510-X DB - PRIME DP - Unbound Medicine ER -