Glucoamylase: structure and mechanisms of action

Starch is an important component of our diet. Enzymatic digestion of ‘crystalline’ starch is important in conveying the health benefits of the resistant starch content of starch-based foods and also in the commercial use of starch as an industrial substrate. In collaboration with Professor B Svensson of the Carlsberg Laboratory, Denmark we have used AFM to study the binding of the starch-degrading enzyme glucoamylase (GA) to starch and, from the nature of the unusual complexes obtained, we have deduced a model for enzymatic digestion of starch.

Glucoamylase is a multi-domain enzyme. The smaller starch-binding domain (SBD) is attached to the larger catalytic domain by a heavily glycosylated linker. Uniquely the SBD contains two binding sites.

To understand how the SBD helps glucoamylase to degrade crystalline starch, we have studied the binding of SBDs, mutated SBDs lacking one of the binding sites, and an inactivated form of the enzyme to the starch polysaccharide amylose. The amylose was prepared as a soluble helical complex.

AFM images (scale bar 100nm) of the amylose show extended linear (helical) structures. The SBDs form rings with amylose and inactivation of either binding site produces linear complexes. The catalytically inactivated enzyme also forms ring-like complexes with amylose.

A model of the amylose-SBD complex, based on the known geometry of the binding sites and the AFM data has been produced. Within the complex the SBDs act as templates for the helical arrangement of the bound amylose molecule.

Complex formation is dominated by the SBD with, in the case of the catalytically inactivated glucoamylase, the catalytic domain decorating the outside of the amylose-SBD ring. The nature of the amylose-SBD complex suggests a role for the SBD. The SBD is considered to recognise and dock onto the ends of amylosic double helices. This locks the otherwise mobile chain ends at the end of the helix in the vicinity of the catalytic domain, facilitating binding and subsequent cleavage.

Further Reading:

Morris VJ, Gunning AP, Faulds CB, Williamson G & Svensson B.
AFM images of complexes between amylose and Aspergillus niger glucoamylase mutants, native and mutant starch binding domains: a model for the action of glucoamylase. Starke 57 (2005) 1-7.

Gunning AP, Giardina TP, Faulds CB, Juge N, Ring SG, Williamson G & Morris VJ.
Surfactant mediated solubilisation of amylose and visualisation by atomic force microscopy. Carbohydrate Polymers 51 (2003) 177-182.

Juge N, Le Gal-Coëffet M-F, Furniss CSM, Gunning AP, Giardina T, Kramhøft, B, Morris VJ, Svensson B & Williamson G.
The starch binding domain of glucoamylase from Aspergillus niger: overview of its structure, function, and role in raw starch hydrolysis. Biologia 57 (2002) 230-245.

Giardina T, Gunning AP, Faulds CB, Juge N, Furniss CSM, Svensson B, Morris VJ & Williamson G.
Influence of the two binding sites of the starch-Binding domain of Aspergillus niger on amylose conformation. J Mol. Biol. 313 (2001) 1151-1161.

Gunning AP, Morris VJ, Kramer GFH, Williamson G, Belshaw NJ & Kanning MW.
Imaging glucoamylase by scanning tunnelling microscopy. The Analyst 119 (1994) 1939-1942.

Kramer GFH, Gunning AP, Morris VJ, Belshaw NJ & Williamson G.
Scanning tunnelling microscopy of Aspergillus niger glucoamylase. J. Chem. Soc. Faraday Trans. 89 (1993) 2595-2602.