title: Glucoamylase: structure/function relationships, and protein engineering.
abstract: Glucoamylases are inverting exo-acting starch hydrolases releasing beta-glucose from the non-reducing ends of starch and related substrates. The majority of glucoamylases are multidomain enzymes consisting of a catalytic domain connected to a starch-binding domain by an O-glycosylated linker region. Three-dimensional structures have been determined of free and inhibitor complexed glucoamylases from Aspergillus awamori var. X100, Aspergillus niger, and Saccharomycopsis fibuligera. The catalytic domain folds as a twisted (alpha/alpha)(6)-barrel with a central funnel-shaped active site, while the starch-binding domain folds as an antiparallel beta-barrel and has two binding sites for starch or beta-cyclodextrin. Certain glucoamylases are widely applied industrially in the manufacture of glucose and fructose syrups. For more than a decade mutational investigations of glucoamylase have addressed fundamental structure/function relationships in the binding and catalytic mechanisms. In parallel, issues of relevance for application have been pursued using protein engineering to improve the industrial properties. The present review focuses on recent findings on the catalytic site, mechanism of action, substrate recognition, the linker region, the multidomain architecture, the engineering of specificity and stability, and roles of individual substrate binding subsites.
authors.affiliation: Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen, Valby, Denmark.
authors.forename: J
authors.initials: J
authors.lastname: Sauer
authors.forename: B W
authors.initials: BW
authors.lastname: Sigurskjold
authors.forename: U
authors.initials: U
authors.lastname: Christensen
authors.forename: T P
authors.initials: TP
authors.lastname: Frandsen
authors.forename: E
authors.initials: E
authors.lastname: Mirgorodskaya
authors.forename: M
authors.initials: M
authors.lastname: Harrison
authors.forename: P
authors.initials: P
authors.lastname: Roepstorff
authors.forename: B
authors.initials: B
authors.lastname: Svensson
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus; D001665:Binding Sites; D002240:Carbohydrate Sequence; D004791:Enzyme Inhibitors / Q000737:chemistry; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry; D008320:Maltose / Q000031:analogs & derivatives / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009844:Oligosaccharides; D008320:Maltose; C009819:maltotetraose; D005087:Glucan 1,4-alpha-Glucosidase