Glucoamylase: structure/function relationships, and protein engineering

Glucoamylases are inverting exo-acting starch hydrolases releasing beta -gl ucose from the non-reducing ends of starch and related substrates. The majo rity of glucoamylases are multidomain enzymes consisting of a catalytic dom ain connected to a starch-binding domain by an O-glycosylated linker region . Three-dimensional structures have been determined of free and inhibitor c omplexed glucoamylases from Aspergillus awamori var. X100, Aspergillus nige r, and Saccharomycopsis fibuligera. The catalytic domain folds as a twisted (alpha/alpha)(6)-barrel with a central funnel-shaped active site, while th e starch-binding domain folds as an antiparallel beta -barrel and has two b inding sites for starch or beta -cyclodextrin. Certain glucoamylases are wi dely applied industrially in the manufacture of glucose and fructose syrups . For more than a decade mutational investigations of glucoamylase have add ressed fundamental structure/function relationships in the binding and cata lytic mechanisms. In parallel, issues of relevance for application have bee n pursued using protein engineering to improve the industrial properties. T he present review focuses on recent findings on the catalytic site, mechani sm of action, substrate recognition, the linker region, the multidomain arc hitecture, the engineering of specificity and stability, and roles of indiv idual substrate binding subsites. (C) 2000 Elsevier Science B.V. All rights reserved.