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

ABSTRACT: Aspergillus fumigatus sterol 14-α 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 μM) and eburicol (K(s), 22.6 μM). Membrane suspensions of AF51A bound to both lanosterol (K(s), 3.1 μM) and eburicol (K(s), 4.1 μM). 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 μM, respectively, in comparison with a K(d) valu

SUBMITTER: Warrilow AG 

PROVIDER: S-EPMC2944604 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature