title: Three-dimensional models of 14α-sterol demethylase (Cyp51A) from Aspergillus lentulus and Aspergillus fumigatus: an insight into differences in voriconazole interaction.
abstract: Aspergillus lentulus, an Aspergillus fumigatus sibling species, is increasingly reported in corticosteroid-treated patients. Its clinical significance is unknown, but the fact that A. lentulus shows reduced antifungal susceptibility, mainly to voriconazole, is of serious concern. Heterologous expression of cyp51A from A. fumigatus and A. lentulus was performed in Saccharomyces cerevisiae to assess differences in the interaction of Cyp51A with the azole drugs. The absence of endogenous ERG11 was efficiently complemented in S. cerevisiae by the expression of either Aspergillus cyp51A allele. There was a marked difference between azole minimum inhibitory concentration (MIC) values of the clones expressing each Aspergillus spp. cyp51A. Saccharomyces cerevisiae clones expressing A. lentulus alleles showed higher MICs to all of the azoles tested, supporting the hypothesis that the intrinsic azole resistance of A. lentulus could be associated with Cyp51A. Homology models of A. fumigatus and A. lentulus Cyp51A protein based on the crystal structure of Cyp51p from Mycobacterium tuberculosis in complex with fluconazole were almost identical owing to their mutual high sequence identity. Molecular dynamics (MD) was applied to both three-dimensional protein models to refine the homology modelling and to explore possible differences in the Cyp51A-voriconazole interaction. After 20ns of MD modelling, some critical differences were observed in the putative closed form adopted by the protein upon voriconazole binding. A closer study of the A. fumigatus and A. lentulus voriconazole putative binding site in Cyp51A suggested that some major differences in the protein's BC loop could differentially affect the lock-up of voriconazole, which in turn could correlate with their different azole susceptibility profiles.
authors.affiliation: Mycology Reference Laboratory, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Carretera Majadahonda-Pozuelo Km 2, 28220 Madrid, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Alcazar-Fuoli
authors.forename: Isabel
authors.initials: I
authors.lastname: Cuesta
authors.forename: Juan L
authors.initials: JL
authors.lastname: Rodriguez-Tudela
authors.forename: Manuel
authors.initials: M
authors.lastname: Cuenca-Estrella
authors.forename: Dominique
authors.initials: D
authors.lastname: Sanglard
authors.forename: Emilia
authors.initials: E
authors.lastname: Mellado
mesh_terms: D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D001230:Aspergillus / Q000187:drug effects / Q000201:enzymology*; D001232:Aspergillus fumigatus / Q000187:drug effects / Q000201:enzymology*; D001665:Binding Sites; D020134:Catalytic Domain; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D025141:Drug Resistance, Fungal; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011743:Pyrimidines / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA; D014230:Triazoles / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D065819:Voriconazole
chemical_list: D000935:Antifungal Agents; D005656:Fungal Proteins; D011743:Pyrimidines; D014230:Triazoles; D003577:Cytochrome P-450 Enzyme System; C465526:cytochrome P-450 CYP51A, Aspergillus; D065819:Voriconazole