Expression, Purification, and Characterization of Aspergillus fumigatus Sterol 14-α Demethylase (CYP51) Isoenzymes A and B

Abstract

Aspergillus fumigatus sterol 14-␣ 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 The sterol pathways of eukaryotes are highly conserved and are part of a larger biosynthetic pathway that includes the formation of dolichols, coenzyme Q, heme A, and isoprenylated proteins. In Saccharomyces cerevisiae, the first step exclusively involved in sterol synthesis is the formation of squalene, with the first sterol intermediate in the pathway being lanosterol, culminating in ergosterol some 15 enzymatic steps later. In fungi, these reactions are governed by individual enzymes, but closer examination of other fungal genome sequences has revealed that there is often duplication (29) and, in some instances, triplicate versions of the same gene (1). We are interested as to why fungi have kept multiple copies of these genes, the roles of the proteins in vivo, and their contribution to both sterol biosynthesis and fungal resistance.,14-␣ Demethylase (CYP51) is an ancestral activity of the cytochrome P450 superfamily, which is also the target of azole antifungals (18). The isolation of CYP51 was initially from Saccharomyces cerevisiae (17), and in the fungal pathogen Aspergillus fumigatus, cytochrome P450 was first observed in 1990 (5, 6). Evidence that alteration of CYP51 activity might contribute to azole resistance first emerged in 1997 (11). In this particular study, we have looked in detail at the biochemical properties of the two CYP51 forms in Aspergillus fumigatus (29) encoded by CYP51A (Afu4g06890) and CYP51B (Afu7g03740). A comparison of the deduced amino acid sequences show 63% identity between them; and both orthologues in A. fumigatus have been shown to act in a compensatory manner in the ergosterol pathway; i.e., neither is essential individually, but a double knockdown is lethal (13). It is postulated that CYP51A may encode the major sterol 14-␣ demethylase activity required for growth on the basis of accumulation of multiple missense mutations linked to azole resistance (31), with CYP51B either being functionally redundant or having an alternative function under particular growth conditions still to be defined. We expressed both proteins in Escherichia coli to investigate their azole binding properties. MATERIALS AND METHODSConstruction of pSPORT AF51A and pSPORT AF51B expression vectors. The coding regions of the A. fumigatus (strain Af293 [http://www.aspergillusgenome .org/gbrowse/afum_af293]) CYP51 isoenzyme A (AF51A) and B (AF51B) genes (ExPASy protein database accession numbers Q4WNT5 and Q96W81, respectively) were synthesized commercially by GeneCust Europe (Dudelange, Luxembourg) in pUC57 with codon optimization for expression in E. coli. NdeI and HindIII sites were included at the 5Ј and 3Ј ends, r...

Structural Insights into Binding of the Antifungal Drug Fluconazole to Saccharomyces cerevisiae Lanosterol 14α-Demethylase

“…L-Histidine was removed from the preparation by washing the enzyme with affinity chromatography medium using a 50-kDa-molecular-mass-cutoff Amicon Ultra-4 centrifugal filter (Millipore). Difference spectra were obtained by titrating affinity-purified ScErg11p6ϫHis and used to determine dissociation constant (K d ) values for the binding to the triazole drugs FLC and VCZ (30,31). Incremental additions of up to 7 l of triazole drug dissolved in dimethyl sulfoxide (DMSO) were added to a 0.5-ml sample of 1 M ScErg11p6ϫHis in affinity chromatography buffer in a 10-mm-pathlength cuvette.…”

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…A. fumigatus is intrinsically resistant to the commonly used azole fluconazole (148), and it was suggested that the specific azole binding properties of A. fumigatus cyp51A and cyp51B may result in the intrinsic fluconazole resistance of this species (618). In 1990, itraconazole became the first azole introduced for the treatment of aspergillosis (146).…”

Azole Drug Import into the Pathogenic Fungus Aspergillus fumigatus

“…Disruption of the ergosterol biosynthesis pathway leads to increased membrane permeability and instability in the fungal cell, which is deleterious to cell growth and replication (6,9). The intrinsic resistance of A. fumigatus to FLC is possibly due to the differential FLC binding affinity to the Cyp51A and Cyp51B proteins (8,10).…”