Fascinating facets of function and structure of amylolytic enzymes of glycoside hydrolase family 13

Birte Svensson, Morten Tovborg Jensen, Haruhide Mori, Kristian Sass Bak-Jensen, Birgit Bønsager, Peter K. Nielsen, Birte Kramhøft, Mette Prægtorius-Ibba, Jane Nøhr, Nathalie Juge, Lionel Greffe, Gary Williamson, Hugues Driguez

Research output: Contribution to journalReview ArticleResearchpeer-review

41 Citations (Scopus)


Glycoside hydrolase family 13 currently comprises enzymes of 28 different specificities, 13 of which are represented by crystal structures. Ligand complex structures are reported for fewer specificities and typically only describe enzyme-sugar interactions for part of the binding area and for α-1,4-linked compounds. Molecular modeling can fill this lack of knowledge and is also supporting the idea that longer substrates apply several binding modes. The double displacement mechanism leading to retention of the substrate anomeric configuration allows production of oligosaccharides by transglycosylation. This is demonstrated using α-amylase 1 isozyme (AMY1) and limit dextrinase from barley. Moreover, the mechanism motivated site-directed mutagenesis of the catalytic nucleophile in an attempt to convert AMY1 into a glycosynthase. Despite correlation of specificity with short sequence motifs in β → α loops of the catalytic (β/α)8-barrel, rational design to alter specificity is not straightforward and the motifs mainly serve to identify target regions for engineering. Here single and dual subsite mutants in AMY1, produced using various mutagenesis strategies, confer changes in i) substrate preference, ii) oligosaccharide product profiles, and iii) degree of multiple attack. Certain hydrolases and transglycosylases have extra N- and C-terminal domains, which mostly are not assigned a function. Aspergillus niger glucoamylase, however, has linker-connected catalytic and starch-binding domains, and served to investigate intramolecular domain communication in starch-hydrolases. Subsequently fusion of the A. niger starch-binding domain with barley AMY1 enhanced the binding affinity and rate of granule hydrolysis, which may be an advantage e.g. in brewing. The presence of proteinaceous inhibitors has been reported for very few GH13 members and generally involves isozyme and species discrimination. Interaction with such naturally-occurring inhibitors has particular relevance in nutrition and for plant defense against pathogens. The sensitivity of barley α-amylase for the endogenous α-amylase/subtilisin inhibitor has been controlled through structure-based mutagenesis.

Original languageEnglish
Pages (from-to)5-19
Number of pages15
JournalBiologia. Section: Cellular and Molecular Biology
Issue numberSUPPL. 11
Publication statusPublished - 1 Dec 2002
Externally publishedYes


  • Barley α-amylase
  • Bond-type specificity
  • Degree of multiple attack
  • N-terminal domains
  • Protein inhibitors
  • Starch binding domain
  • Subsite engineering

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