Two novel, putatively cell wall-associated and glycosylphosphatidylinositol-anchored alpha-glucanotransferase enzymes of Aspergillus niger.
In the genome sequence of Aspergillus niger CBS 513.88, three genes were identified with high similarity to fungal alpha-amylases. The protein sequences derived from these genes were different in two ways from all described fungal alpha-amylases: they were predicted to be glycosylphosphatidylinositol anchored, and some highly conserved amino acids of enzymes in the alpha-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 alpha-(1,4)-glycosidic bonds and at least five anhydroglucose units. The enzymes, designated AgtA and AgtB, produced new alpha-(1,4)-glycosidic bonds and therefore belong to the group of the 4-alpha-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 alpha-(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 alpha-glucans in their cell walls, but not in yeast species lacking cell wall alpha-glucan. Possible roles for these enzymes in the synthesis and/or maintenance of the fungal cell wall are discussed.
van der Kaaij
R M
RM
Centre for Carbohydrate Bioprocessing, TNO-University of Groningen, Haren, The Netherlands.
Yuan
X-L
XL
Franken
A
A
Ram
A F J
AF
Punt
P J
PJ
van der Maarel
M J E C
MJ
Dijkhuizen
L
L
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D002473:Cell Wall / Q000737:chemistry*; D005656:Fungal Proteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D006004:Glycogen Debranching Enzyme System / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D010802:Phylogeny; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D005656:Fungal Proteins; D006004:Glycogen Debranching Enzyme System; D017261:Glycosylphosphatidylinositols; D007527:Isoenzymes; D009844:Oligosaccharides; D011993:Recombinant Fusion Proteins; C021705:maltooligosaccharides; C020569:4 alpha-glucanotransferase