Oligosaccharide and Substrate Binding in the Starch Debranching Enzyme Barley Limit Dextrinase

Marie Sofie Møller, Michael Skovbo Windahl, Lyann Sim, Marie Bøjstrup, Maher Abou Hachem, Ole Hindsgaul, Monica Palcic, Birte Svensson, Anette Henriksen

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    Abstract

    Complete hydrolytic degradation of starch requires hydrolysis of both the α-1,4- and α-1,6-glucosidic bonds in amylopectin. Limit dextrinase (LD) is the only endogenous barley enzyme capable of hydrolyzing the α-1,6-glucosidic bond during seed germination, and impaired LD activity inevitably reduces the maltose and glucose yields from starch degradation. Crystal structures of barley LD and active-site mutants with natural substrates, products and substrate analogues were sought to better understand the facets of LD-substrate interactions that αconfine high activity of LD to branched maltooligosaccharides. For the first time, an intact α-1,6-glucosidically linked substrate spanning the active site of a LD or pullulanase has been trapped and characterized by crystallography. The crystal structure reveals both the branch and main-chain binding sites and is used to suggest a mechanism for nucleophilicity enhancement in the active site. The substrate, product and analogue complexes were further used to outline substrate binding subsites and substrate binding restraints and to suggest a mechanism for avoidance of dual α-1,6- and α-1,4-hydrolytic activity likely to be a biological necessity during starch synthesis.
    Original languageEnglish
    JournalJournal of Molecular Biology
    Volume427
    Issue number6, Part B
    Pages (from-to)1263-1277
    Number of pages15
    ISSN0022-2836
    DOIs
    Publication statusPublished - 2015

    Keywords

    • Pullulanase
    • α-1,6-glucosidase
    • Substrate specificity
    • Thio-oligosaccharide
    • Transglycosylase

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