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

      Fems Microbiology Letters
      Amino Acid Sequence, Azoles, pharmacology, Cloning, Molecular, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System, isolation & purification, physiology, Electrophoresis, Polyacrylamide Gel, Molecular Sequence Data, Oxidoreductases, antagonists & inhibitors, Polymerase Chain Reaction, Sequence Homology, Amino Acid, Spectrophotometry, Sterol 14-Demethylase, Triazoles, Ustilago, enzymology

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          Abstract

          Triadimenol and tebuconazole are potent inhibitors of the sterol 14 alpha-demethylation reaction in fungi which is catalysed by CYP51, a haem-thiolate containing enzyme belonging to the cytochrome P450 monooxygenase superfamily. 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 SDS-PAGE and specific haem content. Catalytic activity was investigated following reconstitution with its respective NADPH cytochrome P450 reductase and proposed endogenous substrate, 24-methylenedihydrolanosterol. Addition of the triadimenol and tebuconazole induced type II spectral changes in the enzyme, with saturation occurring at equimolar azole concentrations. Inhibition of reconstituted activities showed a one-to-one sensitivity of the fungal CYP51 as judged by IC50 values. The implications for fungicide mode of action and treatment are discussed.

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