title: Expression, purification, reconstitution and inhibition of Ustilago maydis sterol 14 alpha-demethylase (CYP51; P450(14DM)).
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.
authors.affiliation: Institute of Biological Sciences, University of Wales Aberystwyth, UK.
authors.forename: D C
authors.initials: DC
authors.lastname: Lamb
authors.forename: D E
authors.initials: DE
authors.lastname: Kelly
authors.forename: N J
authors.initials: NJ
authors.lastname: Manning
authors.forename: D W
authors.initials: DW
authors.lastname: Hollomon
authors.forename: S L
authors.initials: SL
authors.lastname: Kelly
mesh_terms: D000595:Amino Acid Sequence; D001393:Azoles / Q000494:pharmacology; D003001:Cloning, Molecular; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000502:physiology; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D058886:Sterol 14-Demethylase; D014230:Triazoles / Q000494:pharmacology; D014588:Ustilago / Q000201:enzymology*
chemical_list: D001393:Azoles; D065607:Cytochrome P-450 Enzyme Inhibitors; D014230:Triazoles; C032910:triadimefon; C087114:tebuconazole; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase