Journal article
Key aromatic residues at subsites +2 and +3 of glycoside hydrolase family 31 α-glucosidase contribute to recognition of long-chain substrates
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.
Language: | English |
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Year: | 2013 |
Pages: | 329-335 |
ISSN: | 18782434 , 00063002 , 18781454 and 15709639 |
Types: | Journal article |
DOI: | 10.1016/j.bbapap.2012.08.007 |
ORCIDs: | Svensson, Birte |