Starch‐binding domains in the CBM45 family – low‐affinity domains from glucan, water dikinase and α‐amylase involved in plastidial starch metabolism

Mikkel Andreas Glaring, Martin Baumann, Maher Abou Hachem, Hiroyuki Nakai, Natsuko Nakai, Diana Santelia, Bent W. Sigurskjold, Samuel C. Zeeman, Andreas Blennow, Birte Svensson

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    Starch‐binding domains are noncatalytic carbohydrate‐binding modules that mediate binding to granular starch. The starch‐binding domains from the carbohydrate‐binding module family 45 (CBM45, ) are found as N‐terminal tandem repeats in a small number of enzymes, primarily from photosynthesizing organisms. Isolated domains from representatives of each of the two classes of enzyme carrying CBM45‐type domains, the Solanum tuberosumα‐glucan, water dikinase and the Arabidopsis thaliana plastidial α‐amylase 3, were expressed as recombinant proteins and characterized. Differential scanning calorimetry was used to verify the conformational integrity of an isolated CBM45 domain, revealing a surprisingly high thermal stability (Tm of 84.8 °C). The functionality of CBM45 was demonstrated in planta by yellow/green fluorescent protein fusions and transient expression in tobacco leaves. Affinities for starch and soluble cyclodextrin starch mimics were measured by adsorption assays, surface plasmon resonance and isothermal titration calorimetry analyses. The data indicate that CBM45 binds with an affinity of about two orders of magnitude lower than the classical starch‐binding domains from extracellular microbial amylolytic enzymes. This suggests that low‐affinity starch‐binding domains are a recurring feature in plastidial starch metabolism, and supports the hypothesis that reversible binding, effectuated through low‐affinity interaction with starch granules, facilitates dynamic regulation of enzyme activities and, hence, of starch metabolism.
    Original languageEnglish
    JournalF E B S Journal
    Volume278
    Issue number7
    Pages (from-to)1175-1185
    ISSN1742-464X
    DOIs
    Publication statusPublished - 2011

    Keywords

    • a-glucan
    • a-amylase
    • Starch-binding domain
    • Starch metabolism
    • Carbohydrate-binding module
    • Water dikinase

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