Characterization and cilofungin inhibition of solubilized Aspergillus fumigatus (1,3)-beta-D-glucan synthase.

(1,3)-beta-D-Glucan synthase, a major cell wall synthesis enzyme, is the target of antifungal drugs of the lipopeptide class. Aspergillus fumigatus (1,3)-beta-D-glucan synthase was prepared and its activity was measured by incorporation of [14C]glucose from UDP-[U-14C]glucose into an insoluble polymer in the presence of alpha-amylase. Solubilization of the (1,3)-beta-D-glucan synthase was attempted with several detergents, and the maximum percent solubilization was obtained with a polyoxyethylene ether detergent, W-1. Up to 70% of enzyme activity and 50% of total protein were recovered when 1-mg/ml membrane preparations were extracted with 0.045% W-1 at 4 degrees C overnight. Confirmation of the presence of a (1,3)-beta-D-glucose polymer synthesized by this glucan synthase was done by three methods. The first was enzymatic end product degradation by alpha-amylase (no degradation) and beta-glucanase (85 to 95% degradation). The second was gas chromatography-mass spectroscopy analysis of the partially methylated alditol acetate derivatives prepared from total carbohydrate polymers present in the sample. This method identified the presence of (1,3)- and (1,2)-glucosidic linkages. The third was high-performance anion exchange chromatography of radioactive oligosaccharides. This method allowed differentiation of the newly synthesized, radioactive polymers from the contaminating carbohydrates already present in the preparation. The results showed that the polymer synthesized comprised oligosaccharides consistent with beta-(1,3)-linked sugars. Maximal inhibition of the (1,3)-beta-D-glucan synthase by the lipopeptide antifungal agent cilofungin was 80%. Dose-response experiments with this inhibitor showed that the solubilized enzyme was maximally inhibited at a cilofungin concentration of 1.25 microgram/ml and showed <5% inhibition at 0.02 microgram/ml. The apparent K(m) (K(m app)) for the solubilized glucan synthase was 400 +/- 80 microM, and the apparent K(i) (K(i app)) for cilofungin was 0.19 +/- 0.03 microM. Inhibition of A.fumigatus (1,3)-beta-D-glucan synthase with cilofungin was noncompetitive, as it was for the Candida albicans (1,3)-beta-D-glucan synthase.

Oligosaccharide mapping of fungal glucan synthase product by high-performance anion-exchange chromatography.

Crude membrane preparations of fungi contain the enzyme glucan synthase (EC 2.4.1.34) which produces a polymer of glucose linked through 1,3-beta-glycosidic bonds. This polymer is a major structural element of the fungal cell wall. Preparations of glucan synthase are contaminated with the enzyme glycogen synthase (EC 2.4.1.11). Glycogen synthase forms the storage carbohydrate glycogen, a polymer of glucose consisting of mainly 1,4-alpha-glycosidic linkage. Both enzymes utilize uridine diphosphoglucose as substrate. Discrimination of glucan synthase from glycogen synthase activity has relied upon the inclusion of glycogen-degrading enzymes in the crude reactions. The polysaccharide reaction products of glucan synthase assays have been characterized by their susceptibility to enzymatic degradation by various glucanohydrolases. These degradative enzymes are impure and inclusion of appropriate control polysaccharides often leads to ambiguous results. A method for comparative qualitative analysis of polysaccharides formed in fungal glucan synthase reactions has been developed using high-performance anion-exchange chromatography. Using this method, polymers of glucose with 1,3-beta-glycosidic linkage and 1,4-alpha linkage can be readily distinguished. This method has been applied to map oligosaccharides derived by partial acid hydrolysis from fungal glucan synthase reaction products from Candida albicans protoplasts prepared by two different methods.