Purification and characterization of fatty acid synthase from Candida albicans strain 4918 and two derived spontaneous cerulenin-resistant mutants.

Fatty acid synthase from three strains of Candida albicans (parental strain 4918, and two spontaneous cerulenin-resistant mutants, 4918-2 and 4918-10) has been purified and characterized. In all three cases the purification protocol included ammonium sulfate precipitation, fractionation with butyl-Toyopearl, differential centrifugation and sedimentation velocity centrifugation. Inclusion of protease inhibitors, aprotinin, leupeptin and pepstatin was a prerequisite to maximize recoveries. Polyacrylamide gel electrophoresis analysis demonstrated protocol efficacy and showed the apparent molecular mass of the two enzyme sub-units from each strain to be 195 kDa and 210 kDa. The Km (malonyl-CoA) and Vmax of each fatty acid synthase were similar. In contrast, inactivation kinetics of the respective enzymes in the presence of cerulenin showed enzyme activity from both mutants to differ significantly from the parent and from each other. Other experiments suggested in vivo cerulenin resistance of mutant strains is not solely attributable to enzyme alteration.