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Structure-Functional Characterization of Cytochrome P450 Sterol 14α-Demethylase (CYP51B) fromAspergillus fumigatusand Molecular Basis for the Development of Antifungal Drugs

Tatiana Y. Hargrove, Zdzislaw Wawrzak, David Lamb Orcid Logo, F. Peter Guengerich, Galina I. Lepesheva

Journal of Biological Chemistry, Volume: 290, Issue: 39, Pages: 23916 - 23934

Swansea University Author: David Lamb Orcid Logo

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DOI (Published version): 10.1074/jbc.M115.677310

Abstract

Aspergillus fumigatus is the opportunistic fungal pathogen that predominantly affects the immunocompromised population and causes 600,000 deaths/year. The cytochrome P450 51 (CYP51) inhibitor voriconazole is currently the drug of choice, yet the treatment efficiency remains low, calling for rational...

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Published in: Journal of Biological Chemistry
Published: 2015
Online Access: http://www.jbc.org/content/290/39/23916.full.pdf
URI: https://cronfa.swan.ac.uk/Record/cronfa29844
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spelling 2019-05-30T10:39:48.9386403 v2 29844 2016-09-08 Structure-Functional Characterization of Cytochrome P450 Sterol 14α-Demethylase (CYP51B) fromAspergillus fumigatusand Molecular Basis for the Development of Antifungal Drugs 1dc64e55c2c28d107ef7c3db984cccd2 0000-0001-5446-2997 David Lamb David Lamb true false 2016-09-08 BMS Aspergillus fumigatus is the opportunistic fungal pathogen that predominantly affects the immunocompromised population and causes 600,000 deaths/year. The cytochrome P450 51 (CYP51) inhibitor voriconazole is currently the drug of choice, yet the treatment efficiency remains low, calling for rational development of more efficient agents. A. fumigatus has two CYP51 genes, CYP51A and CYP51B, which share 59% amino acid sequence identity. CYP51B is expressed constitutively, whereas gene CYP51A is reported to be inducible. We expressed, purified, and characterized A. fumigatus CYP51B, including determination of its substrate preferences, catalytic parameters, inhibition, and x-ray structure in complexes with voriconazole and the experimental inhibitor (R)-N-(1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VNI). The enzyme demethylated its natural substrate eburicol and the plant CYP51 substrate obtusifoliol at steady-state rates of 17 and 16 min(-1), respectively, but did not metabolize lanosterol, and the topical antifungal drug miconazole was the strongest inhibitor that we identified. The x-ray crystal structures displayed high overall similarity of A. fumigatus CYP51B to CYP51 orthologs from other biological kingdoms but revealed phylum-specific differences relevant to enzyme catalysis and inhibition. The complex with voriconazole provides an explanation for the potency of this small molecule, whereas the complex with VNI outlines a direction for further enhancement of the efficiency of this new inhibitory scaffold to treat humans afflicted with filamentous fungal infections. Journal Article Journal of Biological Chemistry 290 39 23916 23934 cytochrome P450, azole antifungal, crystal structure 25 9 2015 2015-09-25 10.1074/jbc.M115.677310 http://www.jbc.org/content/290/39/23916.full.pdf COLLEGE NANME Biomedical Sciences COLLEGE CODE BMS Swansea University 2019-05-30T10:39:48.9386403 2016-09-08T15:03:16.6174452 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Medicine Tatiana Y. Hargrove 1 Zdzislaw Wawrzak 2 David Lamb 0000-0001-5446-2997 3 F. Peter Guengerich 4 Galina I. Lepesheva 5
title Structure-Functional Characterization of Cytochrome P450 Sterol 14α-Demethylase (CYP51B) fromAspergillus fumigatusand Molecular Basis for the Development of Antifungal Drugs
spellingShingle Structure-Functional Characterization of Cytochrome P450 Sterol 14α-Demethylase (CYP51B) fromAspergillus fumigatusand Molecular Basis for the Development of Antifungal Drugs
David Lamb
title_short Structure-Functional Characterization of Cytochrome P450 Sterol 14α-Demethylase (CYP51B) fromAspergillus fumigatusand Molecular Basis for the Development of Antifungal Drugs
title_full Structure-Functional Characterization of Cytochrome P450 Sterol 14α-Demethylase (CYP51B) fromAspergillus fumigatusand Molecular Basis for the Development of Antifungal Drugs
title_fullStr Structure-Functional Characterization of Cytochrome P450 Sterol 14α-Demethylase (CYP51B) fromAspergillus fumigatusand Molecular Basis for the Development of Antifungal Drugs
title_full_unstemmed Structure-Functional Characterization of Cytochrome P450 Sterol 14α-Demethylase (CYP51B) fromAspergillus fumigatusand Molecular Basis for the Development of Antifungal Drugs
title_sort Structure-Functional Characterization of Cytochrome P450 Sterol 14α-Demethylase (CYP51B) fromAspergillus fumigatusand Molecular Basis for the Development of Antifungal Drugs
author_id_str_mv 1dc64e55c2c28d107ef7c3db984cccd2
author_id_fullname_str_mv 1dc64e55c2c28d107ef7c3db984cccd2_***_David Lamb
author David Lamb
author2 Tatiana Y. Hargrove
Zdzislaw Wawrzak
David Lamb
F. Peter Guengerich
Galina I. Lepesheva
format Journal article
container_title Journal of Biological Chemistry
container_volume 290
container_issue 39
container_start_page 23916
publishDate 2015
institution Swansea University
doi_str_mv 10.1074/jbc.M115.677310
college_str Faculty of Medicine, Health and Life Sciences
hierarchytype
hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Medicine{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Medicine
url http://www.jbc.org/content/290/39/23916.full.pdf
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description Aspergillus fumigatus is the opportunistic fungal pathogen that predominantly affects the immunocompromised population and causes 600,000 deaths/year. The cytochrome P450 51 (CYP51) inhibitor voriconazole is currently the drug of choice, yet the treatment efficiency remains low, calling for rational development of more efficient agents. A. fumigatus has two CYP51 genes, CYP51A and CYP51B, which share 59% amino acid sequence identity. CYP51B is expressed constitutively, whereas gene CYP51A is reported to be inducible. We expressed, purified, and characterized A. fumigatus CYP51B, including determination of its substrate preferences, catalytic parameters, inhibition, and x-ray structure in complexes with voriconazole and the experimental inhibitor (R)-N-(1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VNI). The enzyme demethylated its natural substrate eburicol and the plant CYP51 substrate obtusifoliol at steady-state rates of 17 and 16 min(-1), respectively, but did not metabolize lanosterol, and the topical antifungal drug miconazole was the strongest inhibitor that we identified. The x-ray crystal structures displayed high overall similarity of A. fumigatus CYP51B to CYP51 orthologs from other biological kingdoms but revealed phylum-specific differences relevant to enzyme catalysis and inhibition. The complex with voriconazole provides an explanation for the potency of this small molecule, whereas the complex with VNI outlines a direction for further enhancement of the efficiency of this new inhibitory scaffold to treat humans afflicted with filamentous fungal infections.
published_date 2015-09-25T03:36:22Z
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