ABSTRACT
The essential fungal cell-wall polymer (1,3)beta-glucan is synthesized by the enzyme (1,3)beta-glucan synthase. This enzyme, which is the target of the echinocandin and pneumocandin families of fungicidal antibiotics, is a complex composed of at least two proteins, Rho1p and Fks1p. Homologs of the yeast FKS1 gene have been discovered in numerous fungi, and existing evidence points to, but has not yet proved, Fks1p being the catalytic subunit of (1,3)beta-glucan synthase. We have purified (1,3)beta-glucan synthase from Neurospora crassa approximately 400-fold enrichment and labeled the substrate-binding protein by using a UDP-glucose analog, 5-azido-[beta-(32)P]-UDP-glucose. UDP-glucose-binding proteins were photo-crosslinked to the substrate analog and identified from SDS-PAGE gels by Quadrupole time-of-flight mass spectrometry by sequencing the tryptic peptides. Two plasma membrane proteins were labeled FKS and H(+)-ATPase. These results suggest that FKS appears to be the substrate-binding subunit of (1,3)beta-glucan synthase.
Subject(s)
Fungal Proteins/metabolism , Glucosyltransferases/metabolism , Membrane Proteins/metabolism , Neurospora crassa/metabolism , Schizosaccharomyces pombe Proteins , Uridine Diphosphate Glucose/analogs & derivatives , Uridine Diphosphate Glucose/metabolism , Azides/metabolism , Electrophoresis, Polyacrylamide Gel , Glucosyltransferases/isolation & purification , Mass Spectrometry , Neurospora crassa/enzymology , Proton-Translocating ATPases/metabolismABSTRACT
In our continuing program to identify compounds with antifungal properties, the ethanol extracts of two sponges of the family Plakinidae were found to inhibit the growth of the fungal pathogens Candida albicans and Aspergillus fumigatus. From these organisms three new compounds and five known compounds have been identified. A new 1,2-dioxane ring peroxide acid, 1, has been isolated from the sponge Plakortis halichondrioides along with five known compounds. Two new 1,2-dioxolane peroxide acids, 3 and 4, have been isolated from the sponge Plakinastrella onkodes. The structures were established by interpretation of spectral data. The three new compounds exhibit moderate activity against the fungal pathogen C. albicans with MICs of 5, 1.6, and 1.6 microg/mL respectively, for 1, 3, and 4. Compound 1 also showed in vitro inhibition of the fungal pathogen A. fumigatus with an IC(90) value of 5.6 microg/mL.
