New insight into structure/function relationships in plant alpha-amylase family GH13 members.

Eun-Seong Seo, Joakim Mark Andersen, Morten Munch Nielsen, Malene Bech Vester-Christensen, Camilla Christiansen, Johanne Mørch Jensen, J. A. Mótyán, Š. Janeček, R. Haser, Mikkel Andreas Glaring, A. Blennow, L. Kandra, G. Gyémánt, N. Aghajari, Maher Abou Hachem, Birte Svensson

    Research output: Contribution to journalJournal articleResearchpeer-review


    Two carbohydrate binding surface sites (SBSs) on barley α-amylase 1 (AMY1) of glycoside hydrolase family 13 (GH13) displayed synergy in interactions with starch granules, thus being pivotal for hydrolysis of supramolecular substrates. Mutational analysis showed that SBS1 is more critical for the conversion of starch granules, while SBS2 has higher affinity than SBS1 for β-cyclodextrin (β-CD). Noticeably, the binding preference of β-CD to SBS2 differed distinctly from that of maltooligosaccharides to the catalytic nucleophile mutant D180A AMY1. Binding energy maps at subsites -8 through +4 of the active site indicated remarkably elevated affinity due to the Y380A mutation at SBS2. The high-yield AMY2 expression variant A42P, made it possible to show that Tyr378—corresponding to Tyr380 in AMY1—has a role in interactions with starch granules, but not in β-CD binding. Besides SBSs, dedicated starch binding domains (SBDs) mediate binding to starch granules. SBDs are currently categorised into 9 carbohydrate binding module (CBM) families. A novel CBM20 subfamily encountered in regulatory enzymes possesses characteristically low affinity for β-CD. Although α-amylase is essential for starch mobilisation in germinating barley seeds, efficient degradation requires the concerted action of α-amylase, β-amylase, limit dextrinase (LD) and possibly α-glucosidase. Limit dextrinase (LD) is encoded by a single gene and represents the sole debranching activity during germination. Recent expression of functional LD in Pichia pastoris makes biochemical and biophysical characterisation of this GH13 enzyme possible. An endogenous limit dextrinase inhibitor was cloned and produced recombinantly and demonstrated to have sub-nanomolar affinity for LD.
    Original languageEnglish
    JournalJournal of Applied Glycoscience
    Issue number2
    Pages (from-to)157-162
    Publication statusPublished - 2010


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