Tremellomycetes, a fungal class in the subphylum Agaricomycotina, contain well-known opportunistic and emerging human pathogens. The azole drug fluconazole, used in the treatment of diseases caused by some species of Tremellomycetes, inhibits cytochrome P450 monooxygenase CYP51, an enzyme that converts lanosterol into an essential component of the fungal cell membrane ergosterol. Studies indicate that mutations and over-expression of CYP51 in species of Tremellomycetes are one of the reasons for fluconazole resistance. Moreover, the novel drug, VT-1129, that is in the pipeline is reported to exert its effect by binding and inhibiting CYP51. Despite the importance of CYPs, the CYP repertoire in species of Tremellomycetes has not been reported to date. This study intends to address this research gap. Comprehensive genome-wide CYP analysis revealed the presence of 203 CYPs (excluding 16 pseudo-CYPs) in 23 species of Tremellomycetes that can be grouped into 38 CYP families and 72 CYP subfamilies. Twenty-three CYP families are new and three CYP families (CYP5139, CYP51 and CYP61) were conserved across 23 species of Tremellomycetes. Pathogenic cryptococcal species have 50% fewer CYP genes than non-pathogenic species. The results of this study will serve as reference for future annotation and characterization of CYPs in species of Tremellomycetes.

Cytochrome P450 monooxygenase CYP51 (sterol 14α-demethylase) is a well-known target of the azole drug fluconazole for treating cryptococcosis, a life-threatening fungal infection in immune-compromised patients in poor countries. Studies indicate that mutations in CYP51 confer fluconazole resistance on cryptococcal species. Despite the importance of CYP51 in these species, few studies on the structural analysis of CYP51 and its interactions with different azole drugs have been reported. We therefore performed in silico structural analysis of 11 CYP51s from cryptococcal species and other Tremellomycetes. Interactions of 11 CYP51s with nine ligands (three substrates and six azoles) performed by Rosetta docking using 10,000 combinations for each of the CYP51-ligand complex (11 CYP51s × 9 ligands = 99 complexes) and hierarchical agglomerative clustering were used for selecting the complexes. A web application for visualization of CYP51s’ interactions with ligands was developed (http://bioshell.pl/azoledocking/). The study results indicated that Tremellomycetes CYP51s have a high preference for itraconazole, corroborating the in vitro effectiveness of itraconazole compared to fluconazole. Amino acids interacting with different ligands were found to be conserved across CYP51s, indicating that the procedure employed in this study is accurate and can be automated for studying P450-ligand interactions to cater for the growing number of P450s.