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 antifunga l compounds, and resistance to these drugs and agrochemicals is of sig nificant practical importance, We undertook site-directed mutagenesis of the Candida albicans P45051 heterologously expressed in Saccharomyc es cerevisiae 60 probe a model structure for the enzyme, The change T3 15A 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 subst rate and helped to locate it in the active site, This alteration pertu rbed 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 resist ance to azoles through reduced azole affinity.