Key aromatic residues at subsites +2 and +3 of glycoside hydrolase family 31 α-glucosidase contribute to recognition of long-chain substrates

Takayoshi Tagami, Masayuki Okuyama, Hiroyuki Nakai, Young-Min Kim, Haruhide Mori, Kazunori Taguchi, Birte Svensson, Atsuo Kimura

    Research output: Contribution to journalJournal articleResearchpeer-review

    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 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.
    Original languageEnglish
    JournalB B A - Proteins and Proteomics
    Volume1834
    Issue number1
    Pages (from-to)329-335
    ISSN1570-9639
    DOIs
    Publication statusPublished - 2013

    Keywords

    • α-Glucosidase
    • Glycoside hydrolase family 31
    • Substrate specificity
    • Subsite affinity
    • Aromatic residue

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