title: Structural and Functional Elucidation of Yeast Lanosterol 14α-Demethylase in Complex with Agrochemical Antifungals.
abstract: Azole antifungals, known as demethylase inhibitors (DMIs), target sterol 14α-demethylase (CYP51) in the ergosterol biosynthetic pathway of fungal pathogens of both plants and humans. DMIs remain the treatment of choice in crop protection against a wide range of fungal phytopathogens that have the potential to reduce crop yields and threaten food security. We used a yeast membrane protein expression system to overexpress recombinant hexahistidine-tagged S. cerevisiae lanosterol 14α-demethylase and the Y140F or Y140H mutants of this enzyme as surrogates in order characterize interactions with DMIs. The whole-cell antifungal activity (MIC50 values) of both the R- and S-enantiomers of tebuconazole, prothioconazole (PTZ), prothioconazole-desthio, and oxo-prothioconazole (oxo-PTZ) as well as for fluquinconazole, prochloraz and a racemic mixture of difenoconazole were determined. In vitro binding studies with the affinity purified enzyme were used to show tight type II binding to the yeast enzyme for all compounds tested except PTZ and oxo-PTZ. High resolution X-ray crystal structures of ScErg11p6×His in complex with seven DMIs, including four enantiomers, reveal triazole-mediated coordination of all compounds and the specific orientation of compounds within the relatively hydrophobic binding site. Comparison with CYP51 structures from fungal pathogens including Candida albicans, Candida glabrata and Aspergillus fumigatus provides strong evidence for a highly conserved CYP51 structure including the drug binding site. The structures obtained using S. cerevisiae lanosterol 14α-demethylase in complex with these agrochemicals provide the basis for understanding the impact of mutations on azole susceptibility and a platform for the structure-directed design of the next-generation of DMIs.
authors.affiliation: New Zealand's National School of Pharmacy, University of Otago, Dunedin, New Zealand.
authors.forename: Joel D A
authors.identifier: http://orcid.org/0000-0003-0783-1635
authors.initials: JD
authors.lastname: Tyndall
authors.affiliation: Sir John Walsh Research Institute, New Zealand's National Centre for Dentistry, University of Otago, Dunedin, New Zealand.
authors.forename: Manya
authors.initials: M
authors.lastname: Sabherwal
authors.affiliation: Sir John Walsh Research Institute, New Zealand's National Centre for Dentistry, University of Otago, Dunedin, New Zealand.
authors.forename: Alia A
authors.initials: AA
authors.lastname: Sagatova
authors.affiliation: Sir John Walsh Research Institute, New Zealand's National Centre for Dentistry, University of Otago, Dunedin, New Zealand.|Department of Oral Sciences, New Zealand's National Centre for Dentistry, University of Otago, Dunedin, New Zealand.
authors.forename: Mikhail V
authors.initials: MV
authors.lastname: Keniya
authors.affiliation: Bayer SAS, Division Crop Science, Disease Control Research, Lyon, France.
authors.forename: Jacopo
authors.initials: J
authors.lastname: Negroni
authors.affiliation: Sir John Walsh Research Institute, New Zealand's National Centre for Dentistry, University of Otago, Dunedin, New Zealand.
authors.forename: Rajni K
authors.initials: RK
authors.lastname: Wilson
authors.affiliation: Sir John Walsh Research Institute, New Zealand's National Centre for Dentistry, University of Otago, Dunedin, New Zealand.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Woods
authors.affiliation: Bayer AG, Division Crop Science, Disease Control Research, Monheim, Germany.
authors.forename: Klaus
authors.identifier: http://orcid.org/0000-0002-8199-9180
authors.initials: K
authors.lastname: Tietjen
authors.affiliation: Sir John Walsh Research Institute, New Zealand's National Centre for Dentistry, University of Otago, Dunedin, New Zealand.|Department of Oral Sciences, New Zealand's National Centre for Dentistry, University of Otago, Dunedin, New Zealand.
authors.forename: Brian C
authors.initials: BC
authors.lastname: Monk
mesh_terms: D058888:14-alpha Demethylase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D016573:Agrochemicals / Q000737:chemistry* / Q000494:pharmacology; D000935:Antifungal Agents / Q000737:chemistry* / Q000494:pharmacology; D001393:Azoles / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites; D020134:Catalytic Domain; D008826:Microbial Sensitivity Tests; D008968:Molecular Conformation; D015394:Molecular Structure; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology; D058886:Sterol 14-Demethylase / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D015003:Yeasts / Q000187:drug effects / Q000201:enzymology*
chemical_list: D058888:14-alpha Demethylase Inhibitors; D016573:Agrochemicals; D000935:Antifungal Agents; D001393:Azoles; D058886:Sterol 14-Demethylase