Probing the Active Site of Cytochrome P450 2B1:  Metabolism of 7-Alkoxycoumarins by the Wild Type and Five Site-Directed Mutants

Kobayashi, Yasuna; Fang, Xiaojun; Szklarz, Grazyna D.; Halpert, James R.

doi:10.1021/bi9731450

Cited by 31 publications

(29 citation statements)

References 32 publications

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“…The resulting metabolite profile was remarkably similar to that of CYP2B1. Kobayashi et al (1998) observed that substitution of Val-363 with leucine in CYP2B1 almost abolished the production of 7-hydroxycoumarin and favored the production of 7-(3-hydroxybutoxy)coumarin. Our data revealed that the special metabolite ratio observed for CYP2B1 V363L is characteristic for CYP2B6.…”

Section: Discussionmentioning

confidence: 97%

“…In several studies, 7-butoxycoumarin was found to be very informative as an active-site probe for CYP2B1, as this substrate can undergo regioselective hydroxylation on the side chain (Kobayashi et al, 1998;. Therefore, 7-butoxycoumarin allowed us to analyze the architecture of the active sites of CYP2B6 and CYP2E1 more carefully and was used at a concentration according to .…”

Section: Downloaded Frommentioning

confidence: 99%

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Analysis of Differential Substrate Selectivities of CYP2B6 and CYP2E1 by Site-Directed Mutagenesis and Molecular Modeling

Spatzenegger

Liu

Wang

et al. 2003

J Pharmacol Exp Ther

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Human CYP2B6 and CYP2E1 were used to investigate the extent to which differential substrate selectivities between cytochrome P450 subfamilies reflect differences in active-site residues as opposed to distinct arrangement of the backbone of the enzymes. Reciprocal CYP2B6 and CYP2E1 mutants at active-site positions 103, 209, 294, 363, 367, and 477 (numbering according to CYP2B6) were characterized using the CYP2B6-selective substrate 7-ethoxy-4-trifluoromethylcoumarin, the CYP2E1-selective substrate p-nitrophenol, and the common substrates 7-ethoxycoumarin, 7-butoxycoumarin, and arachidonic acid. This report is the first to study the active site of CYP2E1 by systematic site-directed mutagenesis. One of the most intriguing findings was that substitution of CYP2E1 Phe-477 with valine from CYP2B6 resulted in significant 7-ethoxy-4-trifluoromethylcoumarin deethylation. Use of threedimensional models of CYP2B6 and CYP2E1 based on the crystal structure of CYP2C5 suggested that deethylation of 7-ethoxy-4-trifluoromethylcoumarin by CYP2E1 is impeded by van der Waals overlaps with the side chain of Phe-477. Interestingly, none of the CYP2B6 mutants acquired enhanced ability to hydroxylate p-nitrophenol. Substitution of residue 363 in CYP2E1 and CYP2B6 resulted in significant alterations of the metabolite profile for the side chain hydroxylation of 7-butoxycoumarin. Probing of CYP2E1 mutants with arachidonic acid indicated that residues Leu-209 and Phe-477 are critical for substrate orientation in the active site. Overall, the study revealed that differences in the side chains of active-site residues are partially responsible for differential substrate selectivities across cytochrome P450 subfamilies. However, the relative importance of active-site residues appears to be dependent on the structural similarity of the compound to other substrates of the enzyme.

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Section: Discussionmentioning

confidence: 97%

Section: Downloaded Frommentioning

confidence: 99%

Analysis of Differential Substrate Selectivities of CYP2B6 and CYP2E1 by Site-Directed Mutagenesis and Molecular Modeling

Spatzenegger

Liu

Wang

et al. 2003

J Pharmacol Exp Ther

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“…Modeling and experimental techniques were used to investigate the effect of the chain length on the metabolism of a series of 7-alkoxycoumarins by P450 2B1 and its mutants (Kobayashi et al, 1998). The ability of the wild-type enzyme to oxidize a specific compound was found to depend upon the length of the alkyl chain.…”

Section: Liu Et Almentioning

confidence: 99%

Characterization of Substrate Binding to Cytochrome P450 1A1 Using Molecular Modeling and Kinetic Analyses: Case of Residue 382

Liu

Ericksen²,

Besspiata³

et al. 2003

Drug Metab Dispos

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This article is available online at http://dmd.aspetjournals.org ABSTRACT:Key residue Val-382 in P450 1A1 has been predicted to interact with the alkoxy chain of resorufin derivatives. Therefore, we undertook a detailed analysis of substrate mobility in the active site of the P450 1A1 homology model and assessed the effect of mutations at position 382. Dynamic trajectories of 7-methoxy-, 7-ethoxy-, and 7-pentoxyresorufin indicated that 7-ethoxyresorufin would be oxidized most efficiently by the wild-type enzyme. The Val-3823Ala mutation would increase the O-dealkylation of 7-pentoxyresorufin but decrease the oxidation of other substrates. In the case of the V382L mutant, the large bulk of Leu would block alkoxyresorufins from productive binding orientations leading to lowered activities. Binding free energy calculations for three substrates with 1A1 WT and two mutants indicated that binding constants would be similar for all enzyme-substrate combinations.Modeling predictions were tested experimentally. The plasmid containing the cDNA for human P450 1A1 modified for bacterial expression was altered to include a C-terminal PCR-generated six histidine domain to facilitate enzyme purification. The V382A and V382L mutants were constructed by site-directed mutagenesis and Escherichia coli-expressed enzymes purified using Ni-NTA affinity chromatography. The activity of the WT 1A1 was highest toward 7-ethoxyresorufin and lowest toward 7-pentoxyresorufin. Both mutants displayed a decrease in V max with 7-methoxy-and 7-ethoxyresorufin, whereas for the V382A mutant, V max with 7-pentoxyresorufin was increased. No significant changes in K m were observed relative to the wild-type enzyme. The experimental results are thus in good agreement with modeling predictions.

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“…In addition, site-directed mutagenesis was crucial to understand the structural and functional role of residues in the active site, access channel, and a recently observed peripheral pocket (Kobayashi et al, 1998;Hernandez et al, 2006;Jang et al, 2014Jang et al, , 2015. Our multifaceted approach has revealed how movement of secondary structure elements, such as the F-G helices and I helix, and reorientation of active-site residues, such as F206 and F297, allow CYP2B enzymes to bind compounds as small as a-pinene (molecular weight 5 136 g/mol) and as large as two molecules of amlodipine (molecular weight 5 409 g/mol) with high affinity.…”

Section: Introductionmentioning

confidence: 99%

Structure-Function Analysis of Mammalian CYP2B Enzymes Using 7-Substituted Coumarin Derivatives as Probes: Utility of Crystal Structures and Molecular Modeling in Understanding Xenobiotic Metabolism

et al. 2016

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Crystal structures of CYP2B35 and CYP2B37 from the desert woodrat were solved in complex with 4-(4-chlorophenyl)imidazole (4-CPI). The closed conformation of CYP2B35 contained two molecules of 4-CPI within the active site, whereas the CYP2B37 structure demonstrated an open conformation with three 4-CPI molecules, one within the active site and the other two in the substrate access channel. To probe structurefunction relationships of CYP2B35, CYP2B37, and the related CYP2B36, we tested the O-dealkylation of three series of related substrates-namely, 7-alkoxycoumarins, 7-alkoxy-4-(trifluoromethyl)coumarins, and 7-alkoxy-4-methylcoumarinswith a C1-C7 side chain. CYP2B35 showed the highest catalytic efficiency (k cat /K M ) with 7-heptoxycoumarin as a substrate, followed by 7-hexoxycoumarin. In contrast, CYP2B37 showed the highest catalytic efficiency with 7-ethoxy-4-(trifluoromethyl) coumarin (7-EFC), followed by 7-methoxy-4-(trifluoromethyl) coumarin (7-MFC). CYP2B35 had no dealkylation activity with 7-MFC or 7-EFC. Furthermore, the new CYP2B-4-CPI-bound structures were used as templates for docking the 7-substituted coumarin derivatives, which revealed orientations consistent with the functional studies. In addition, the observation of multiple -Cl and -NH-p interactions of 4-CPI with the aromatic side chains in the CYP2B35 and CYP2B37 structures provides insight into the influence of such functional groups on CYP2B ligand binding affinity and specificity. To conclude, structural, computational, and functional analysis revealed striking differences between the active sites of CYP2B35 and CYP2B37 that will aid in the elucidation of new structure-activity relationships.

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Probing the Active Site of Cytochrome P450 2B1: Metabolism of 7-Alkoxycoumarins by the Wild Type and Five Site-Directed Mutants

Cited by 31 publications

References 32 publications

Analysis of Differential Substrate Selectivities of CYP2B6 and CYP2E1 by Site-Directed Mutagenesis and Molecular Modeling

Analysis of Differential Substrate Selectivities of CYP2B6 and CYP2E1 by Site-Directed Mutagenesis and Molecular Modeling

Characterization of Substrate Binding to Cytochrome P450 1A1 Using Molecular Modeling and Kinetic Analyses: Case of Residue 382

Structure-Function Analysis of Mammalian CYP2B Enzymes Using 7-Substituted Coumarin Derivatives as Probes: Utility of Crystal Structures and Molecular Modeling in Understanding Xenobiotic Metabolism

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