title: Key aromatic residues at subsites +2 and +3 of glycoside hydrolase family 31 α-glucosidase contribute to recognition of long-chain substrates.
abstract: 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 k(cat)/K(m) 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 k(cat)/K(m) values for substrates longer than maltose. Providing a phenylalanine residue at a similar position in ANG (T228F) altered the k(cat)/K(m) values for maltooligosaccharides compared with wild-type ANG, i.e., the mutant enzyme showed the highest k(cat)/K(m) value of 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.
authors: Takayoshi
authors: T
authors: Tagami
authors: Masayuki
authors: M
authors: Okuyama
authors: Hiroyuki
authors: H
authors: Nakai
authors: Young-Min
authors: YM
authors: Kim
authors: Haruhide
authors: H
authors: Mori
authors: Kazunori
authors: K
authors: Taguchi
authors: Birte
authors: B
authors: Svensson
authors: Atsuo
authors: A
authors: Kimura
mesh_terms: D019943:Amino Acid Substitution; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008320:Maltose / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D009844:Oligosaccharides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D009844:Oligosaccharides; D010649:Phenylalanine; D008320:Maltose; D000520:alpha-Glucosidases