title: Catalytic properties of the Gas family β-(1,3)-glucanosyltransferases active in fungal cell-wall biogenesis as determined by a novel fluorescent assay.
abstract: BGTs [β-(1,3)-glucanosyltransglycosylases; EC 2.4.1.-] of the GH72 (family 72 of glycosylhydrolases) are GPI (glycosylphosphatidylinositol)-anchored proteins that play an important role in the biogenesis of fungal cell walls. They randomly cleave glycosidic linkages in β-(1,3)-glucan chains and ligate the polysaccharide portions containing newly formed reducing ends to C(3)(OH) at non-reducing ends of other β-(1,3)-glucan molecules. We have developed a sensitive fluorescence-based method for the assay of transglycosylating activity of GH72 enzymes. In the new assay, laminarin [β-(1,3)-glucan] is used as the glucanosyl donor and LamOS (laminarioligosaccharides) fluorescently labelled with SR (sulforhodamine) serve as the acceptors. The new fluorescent assay was employed for partial biochemical characterization of the heterologously expressed Gas family proteins from the yeast Saccharomyces cerevisiae. All the Gas enzymes specifically used laminarin as the glucanosyl donor and a SR-LamOS of DP (degree of polymerization) ≥5 as the acceptors. Gas proteins expressed in distinct stages of the yeast life cycle showed differences in their pH optima. Gas1p and Gas5p, which are expressed during vegetative growth, had the highest activity at pH 4.5 and 3.5 respectively, whereas the sporulation-specific Gas2p and Gas4p were most active between pH 5 and 6. The novel fluorescent assay provides a suitable tool for the screening of potential glucanosyltransferases or their inhibitors.
authors.affiliation: Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Bratislava, Slovakia.
authors.forename: Marián
authors.initials: M
authors.lastname: Mazáň
authors.forename: Enrico
authors.initials: E
authors.lastname: Ragni
authors.forename: Laura
authors.initials: L
authors.lastname: Popolo
authors.forename: Vladimír
authors.initials: V
authors.lastname: Farkaš
mesh_terms: D055162:Biocatalysis; D002473:Cell Wall / Q000378:metabolism*; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
chemical_list: D005456:Fluorescent Dyes; D009844:Oligosaccharides; C117939:1,3-beta-glucanosyltransferase; D004693:Glucan Endo-1,3-beta-D-Glucosidase