Biologia, Bratislava, 57/Suppl. 11: 5-19, 2002.

ISSN 0006-3088 (Biologia).

 

Keynote Review

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

 

Birte Svensson1*, Morten Tovborg Jensen1, Haruhide Mori1,2, Kristian Sass Bak-Jensen1, Birgit Boensager1, Peter K. Nielsen1, Birte Kramhoft1, Mette Praetorius-Ibba1, Jane Nohr3, Nathalie Juge4, Lionel Greffe5, Gary Williamson4 & Hugues Driguez5

1 Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen, Denmark; tel.: ++ 45 3327 5345, fax: ++ 45 3327 4708, e-mail: bis@crc.dk

2 Permanent address: Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

3 Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark

4 Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK

5 Centre de Recherche sur les Macromolécules Végétales, CNRS (affiliated with Université Joseph Fourier), BP 53, F-38401 Grenoble Cedex 09, France

* corresponding author

Received: November 12, 2001 / Accepted: February 12, 2002

 

Abstract

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 a-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 a-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 b®a loops of the catalytic (b/a)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 a-amylase for the endogenous a-amylase/subtilisin inhibitor has been controlled through structure-based mutagenesis.

 

Key words: barley alpha-amylase, bond-type specificity, subsite engineering, degree of multiple attack, N-terminal domains, starch binding domain, protein inhibitors.