Expression, purification, reconstitution and inhibition of Ustilago maydissterol 14 alpha-demethylase (CYP51; P450(14DM))

Triadimenol and tebuconazole are potent inhibitors of the sterol 14 alpha-d emethylation reaction in fungi which is catalysed by CYP51. a haem-thiolate containing enzyme belonging to the cytochrome P450 monooxygenase superfami ly. Using CYP51 from the phytopathogen Ustilago maydis, a comparison of the sensitivity of the fungal enzyme to triadimenol and tebuconazole has been carried out. U, maydis CYP51 was purified to homogeneity as determined by S DS-PAGE and specific haem content. Catalytic activity was investigated foll owing reconstitution with its respective NADPH cytochrome P450 reductase an d proposed endogenous substrate, 24-methylenedihydrolanosterol. Addition of the triadimenol and tebuconazole induced type II spectral changes in the e nzyme, with saturation occurring at equimolar azole concentrations. Inhibit ion of reconstituted activities showed a one-to-one sensitivity of the fung al CYP51 as judged by IC50 values. The implications for fungicide mode of a ction and treatment are discussed. (C) 1998 Federation of European Microbio logical Societies. Published by Elsevier Science B.V. All rights reserved.