Catalytic properties of the Gas family β-(1,3)-glucanosyltransferases active in fungal cell wall biogenesis as determined by a novel fluorescent assay
Résumé
Glucanosyltransferases (β-1,3-glucanosyl transglycosylases, EC 2.4.1.-) of the glycosyl hydrolase family GH72 are glycosyl phosphatidylinositol-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 C3(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 laminarioligosaccharides fluorescently-labeled with sulforhodamine (SR-laminarioligosaccharides) 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 SR-laminarioligosaccharides of DP ≥ 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 glucanosyl transferases or their inhibitors.
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