Two Novel, Putatively Cell Wall-Associated and Glycosylphosphatidylinositol-Anchored

In the genome sequence of Aspergillus niger CBS 513.88, three genes were identified with high similarity to fungal -amylases. The protein sequences derived from these genes were different in two ways from all described fungal -amylases: they were predicted to be glycosylphosphatidylinositol anchored, and some highly conserved amino acids of enzymes in the -amylase family were absent. We expressed two of these enzymes in a suitable A. niger strain and characterized the purified proteins. Both enzymes showed transglycosylation activity on donor substrates with -(1,4)-glycosidic bonds and at least five anhydroglucose units. The enzymes, designated AgtA and AgtB, produced new -(1,4)-glycosidic bonds and therefore belong to the group of the 4--glucanotransferases (EC 2.4.1.25). Their reaction products reached a degree of polymerization of at least 30. Maltose and larger maltooligosaccharides were the most efficient acceptor substrates, although AgtA also used small nigerooligosaccharides containing -(1,3)-glycosidic bonds as acceptor substrate. An agtA knockout of A. niger showed an increased susceptibility towards the cell wall-disrupting compound calcofluor white, indicating a cell wall integrity defect in this strain. Homologues of AgtA and AgtB are present in other fungal species with -glucans in their cell walls, but not in yeast species lacking cell wall -glucan. Possible roles for these enzymes in the synthesis and/or maintenance of the fungal cell wall are discussed