Interaction of tetraconazole and its enantiomers with cytochrome P450 from Ustilago maydis

Azole antifungal agents are known to inhibit 14α‐demethylation during ergosterol biosynthesis which is mediated by a cytochrome P450. Tetraconazole and its enantiomers have been evaluated for their activity in Ustilago maydis sporidial growth inhibition and for their interaction with microsomal P450 isolated from this pathogenic fungus. The methodology developed for isolation of microsomes exhibiting a P450 peak in reduced carbon monoxide difference spectra required the preparation and gentle lysis of protoplasts. Using this fraction, tetraconazole was observed to bind to U. maydis P450 producing a Type II spectrum indicative of coordination between the triazole N‐4 and the haem Fe3+. Carbon monoxide displacement of azole bound to P450 was monitored and was found to reflect the relative activity of the enantiomers when treating cells. The results imply that the activity of the enantiomers is due to their affinity with the target enzyme and that the orientation of the substituents on the chiral carbon is important in binding to the sterol 14α‐demethylase of this phytopathogen.