SOLUTION STRUCTURE OF THE GRANULAR STARCH BINDING DOMAIN OF ASPERGILLUS-NIGER GLUCOAMYLASE BOUND TO BETA-CYCLODEXTRIN

Background: Carbohydrate-binding domains are usually small and physica lly separate from the catalytic domains of hydrolytic enzymes. Glucoam ylase 1 (G1) from Aspergillus niger, an enzyme used widely in the food and brewing industries, contains a granular starch binding domain (SB D) which is separated from the catalytic domain by a semi-rigid linker . The aim of this study was to determine how the SBD binds to starch, and thereby more generally to throw light on the role of carbohydrate- binding domains in the hydrolysis of insoluble polysaccharides. Result s: The solution structure of the SBD of A. niger G1 bound to beta-cycl odextrin (beta CD), a cyclic starch analogue, shows that the well-defi ned beta-sheet structure seen in the free SBD is maintained in the SBD -beta CD complex. The main differences between the free and bound stat es of the SBD are observed in loop regions, in or near the two starch- binding sites. The two binding sites, each of which binds one molecule of beta CD, are structurally different. Binding site 1 is small and a ccessible, and its structure changes very little upon ligand binding. Site 2 is longer and undergoes a significant structural change on bind ing. Part of this site comprises a flexible loop, which appears to all ow the SBD to bind to starch strands in a range of orientations. Concl usions: The two starch-binding sites of the SBD probably differ functi onally as well as structurally; site 1 probably acts as the initial st arch recognition site, whereas site 2 is involved in specific recognit ion of appropriate regions of starch. The two starch strands are bound at approximately 90 degrees to each other. This may be functionally i mportant, as it may force starch strands apart thus increasing the hyd rolyzable surface, or alternatively it may localize the enzyme to nonc rystalline (more hydrolyzable) areas of starch. The region of the SBD where the linker to the catalytic domain is attached is flexible, allo wing the catalytic site to access a large surface area of the starch g ranules.