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

    Cite this

    Møller, Marie Sofie ; Windahl, Michael Skovbo ; Sim, Lyann ; Bøjstrup, Marie ; Abou Hachem, Maher ; Hindsgaul, Ole ; Palcic, Monica ; Svensson, Birte ; Henriksen, Anette. / Oligosaccharide and Substrate Binding in the Starch Debranching Enzyme Barley Limit Dextrinase. In: Journal of Molecular Biology. 2015 ; Vol. 427, No. 6, Part B. pp. 1263-1277.
    @article{411eab729f5e4265a41f9cf60a441f60,
    title = "Oligosaccharide and Substrate Binding in the Starch Debranching Enzyme Barley Limit Dextrinase",
    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.",
    keywords = "Pullulanase, α-1,6-glucosidase, Substrate specificity, Thio-oligosaccharide, Transglycosylase",
    author = "M{\o}ller, {Marie Sofie} and Windahl, {Michael Skovbo} and Lyann Sim and Marie B{\o}jstrup and {Abou Hachem}, Maher and Ole Hindsgaul and Monica Palcic and Birte Svensson and Anette Henriksen",
    year = "2015",
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    volume = "427",
    pages = "1263--1277",
    journal = "Journal of Molecular Biology",
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    }

    Oligosaccharide and Substrate Binding in the Starch Debranching Enzyme Barley Limit Dextrinase. / Møller, Marie Sofie; Windahl, Michael Skovbo; Sim, Lyann; Bøjstrup, Marie; Abou Hachem, Maher ; Hindsgaul, Ole; Palcic, Monica; Svensson, Birte; Henriksen, Anette.

    In: Journal of Molecular Biology, Vol. 427, No. 6, Part B, 2015, p. 1263-1277.

    Research output: Contribution to journalJournal articleResearchpeer-review

    TY - JOUR

    T1 - Oligosaccharide and Substrate Binding in the Starch Debranching Enzyme Barley Limit Dextrinase

    AU - Møller, Marie Sofie

    AU - Windahl, Michael Skovbo

    AU - Sim, Lyann

    AU - Bøjstrup, Marie

    AU - Abou Hachem, Maher

    AU - Hindsgaul, Ole

    AU - Palcic, Monica

    AU - Svensson, Birte

    AU - Henriksen, Anette

    PY - 2015

    Y1 - 2015

    N2 - 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.

    AB - 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.

    KW - Pullulanase

    KW - α-1,6-glucosidase

    KW - Substrate specificity

    KW - Thio-oligosaccharide

    KW - Transglycosylase

    U2 - 10.1016/j.jmb.2014.12.019

    DO - 10.1016/j.jmb.2014.12.019

    M3 - Journal article

    VL - 427

    SP - 1263

    EP - 1277

    JO - Journal of Molecular Biology

    JF - Journal of Molecular Biology

    SN - 0022-2836

    IS - 6, Part B

    ER -