The mutation T315A in Candida albicans sterol 14 alpha-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity

Sterol 14 alpha-demethylase (P45051) is the target for azole antifungal compounds, and resistance to these drugs and agrochemicals is of significant practical importance, We undertook site-directed mutagenesis of the Candida albicans P45051 heterologously expressed in Saccharomyces cerevisiae 60 probe a model structure for the enzyme, The change T315A reduced enzyme activity 2-fold as predicted for the removal of the residue that formed a hydrogen bond with the 3-OH of the sterol substrate and helped to locate it in the active site, This alteration perturbed the heme environment, causing an altered reduced carbon monoxide difference spectrum with a maximum at 445 mn. The changes also reduced the affinity of the enzyme for the azole antifungals ketoconazole and fluconazole and after expression induced by galactose caused 45-fold azole resistance in transformants of S, cerevisiae. This is the first example of a single base change in the target enzyme conferring resistance to azoles through reduced azole affinity.