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.
Highlights
Since the introduction in 1973 of the imidazole antifungal agent enilconazole and the triazole triadimefon, multiple generations of the azole antifungals [1,2,3,4] have underpinned global food security by preventing or treating a wide range of diseases in plants caused by fungal pathogens [5]
To better understand how antifungal CYP51 inhibitors of phytopathogens work, we have determined seven X-ray crystal structures using the surrogate enzyme ScErg11p6×His. These complexes reveal the specific binding orientation of individual enantiomers of two azole agrochemicals that are administered in the field as racemic mixtures (S/R-TBZ and S/R-DPZ) along with FQZ, PRZ and the racemic mixture of four stereoisomers of DFC
Biochemical studies have measured azole binding to the affinity purified enzyme and identified S-TBZ and S-DPZ as the more active enantiomers within the set of molecules tested against S. cerevisiae cells
Summary
Since the introduction in 1973 of the imidazole antifungal agent enilconazole (imazalil, chloramazole) and the triazole triadimefon, multiple generations of the azole antifungals [1,2,3,4] have underpinned global food security by preventing or treating a wide range of diseases in plants caused by fungal pathogens (for review see Parker et al.) [5]. Azoles are routinely used to treat diseases caused by phytopathogenic fungi including eyespot disease and powdery mildew in wheat and barley, black rust, headblight and septoria leaf blotch in wheat, leaf scald in cereals, leaf spot in sugar beet, powdery mildew on grapes, black sigatoka in bananas, apple scab, and mold on citrus fruit as well as the production of toxins. Without agrochemical intervention such diseases can substantially reduce crop yields, wipe out important crops, or result in crop spoilage due to rots or mycotoxins such as aflotoxins in peanuts. In a large study profiling the toxicities of environmental chemicals, it was found that the reproductive toxicity caused by individual azoles varies widely [17]
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