1 Department of Systems Biology, Technical University of Denmark2 Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark3 Hokkaido University4 Memuro Upland Farming Research Division5 Enzyme and Protein Chemistry, Department of Biotechnology and Biomedicine, Technical University of Denmark6 Hokkaido University
Glycoside hydrolase family 31 α-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger α-glucosidase (ANG) is specific for short-chain substrates with the highest kcat/Km for maltotriose, while sugar beet α-glucosidase (SBG) prefers long-chain substrates and soluble starch. Multiple sequence alignment of 31AGs indicated a high degree of diversity at the long loop (N-loop), which forms one wall of the active pocket. Mutations of Phe236 in the N-loop of SBG (F236A/S) decreased kcat/Km values for substrates longer than maltose. Providing a phenylalanine residue at a similar position in ANG (T228F) altered the kcat/Km values for maltooligosaccharides compared with wild-type ANG, i.e., the mutant enzyme showed the highest kcat/Km value for maltotetraose. Subsite affinity analysis indicated that modification of subsite affinities at +2 and +3 caused alterations of substrate specificity in the mutant enzymes. These results indicated that the aromatic residue in the N-loop contributes to determining the chain-length specificity of 31AGs.
B B a - Proteins and Proteomics, 2013, Vol 1834, Issue 1, p. 329-335