Expression, purification, and characterization of Aspergillus fumigatus sterol 14-alpha demethylase (CYP51) isoenzymes A and B.

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Warrilow AG, Melo N, Martel CM, Parker JE, Nes WD, Kelly SL, Kelly DE

Expression, purification, and characterization of Aspergillus fumigatus sterol 14-alpha demethylase (CYP51) isoenzymes A and B.

Antimicrob Agents Chemother. 2010 Oct;54(10):4225-34. doi: 10.1128/AAC.00316-10. Epub 2010 Jul 26.

PubMed ID

20660663 [ View in PubMed

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Abstract

Aspergillus fumigatus sterol 14-alpha demethylase (CYP51) isoenzymes A (AF51A) and B (AF51B) were expressed in Escherichia coli and purified. The dithionite-reduced CO-P450 complex for AF51A was unstable, rapidly denaturing to inactive P420, in marked contrast to AF51B, where the CO-P450 complex was stable. Type I substrate binding spectra were obtained with purified AF51B using lanosterol (K(s), 8.6 muM) and eburicol (K(s), 22.6 muM). Membrane suspensions of AF51A bound to both lanosterol (K(s), 3.1 muM) and eburicol (K(s), 4.1 muM). The binding of azoles, with the exception of fluconazole, to AF51B was tight, with the K(d) (dissociation constant) values for clotrimazole, itraconazole, posaconazole, and voriconazole being 0.21, 0.06, 0.12, and 0.42 muM, respectively, in comparison with a K(d) value of 4 muM for fluconazole. Characteristic type II azole binding spectra were obtained with AF51B, whereas an additional trough and a blue-shifted spectral peak were present in AF51A binding spectra for all azoles except clotrimazole. This suggests two distinct azole binding conformations within the heme prosthetic group of AF51A. All five azoles bound relatively weakly to AF51A, with K(d) values ranging from 1 muM for itraconazole to 11.9 muM for fluconazole. The azole binding properties of purified AF51A and AF51B suggest an explanation for the intrinsic azole (fluconazole) resistance observed in Aspergillus fumigatus.

DrugBank Data that Cites this Article

Drug Targets

Drug	Target	Kind	Organism	Pharmacological Action	Actions
Posaconazole	Cytochrome P450 51	Protein	Yeast	Yes	Antagonist	Details