Structural Studies of Three Glycosidases

• Main Author: Larsson, Anna
• Format: Doctoral Thesis
• Language: English
• Published: Uppsala universitet, Institutionen för cell- och molekylärbiologi 2006
• Subjects: Cell and molecular biology; glycosidase; dextranase; mannanase; endoglucanase; SeMet; MAD; crystal structure; Cell- och molekylärbiologi
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6339; http://nbn-resolving.de/urn:isbn:91-554-6461-0

Glycosidases hydrolyse the glycosidic bond in carbohydrates. Structural studies of three glycosidases with different substrate specificities are presented in this work. Dextranase catalyzes the hydrolysis of α-1,6-glycosidic linkage in dextran polymers. The structure of dextranase, Dex49A, from Penicillium minioluteum was solved in the apo-enzyme (1.8 Å resolution) and product-bound (1.65 Å resolution) forms. The main domain of the enzyme is a right-handed β-helix, which is connected to a β-sandwich domain at the N-terminus. Using NMR spectroscopy the reaction course was shown to occur with net inversion at the anomeric carbon. A new clan is suggested that links glycoside hydrolase (GH) families 28 and 49. Endo-β-1,4-D-mannanase catalyzes the depolymerization of β-1,4-mannan polymers. The structure of endo-1,4-β-mannanase Man5A from blue mussel Mytilus edulis has been determined at 1.6 Å resolution. Kinetic analysis of Man5A revealed that the enzyme requires at least 6 subsites for efficient hydrolysis. The architecture of the catalytic cleft differs significantly from other GH 5 enzyme structures. We therefore suggest that Man5A represents a new subfamily in GH 5. Both the Dex49A and the Man5A structures were determined by multiple-wavelength anomalous diffraction using the selenium K-edge with selenomethionyl enzymes expressed in the yeast Pichia pastoris. Endoglucanase Cel6A from Thermobifida fusca hydrolyzes the β-1,4 linkages in cellulose. The structure of the catalytic domain of Cel6A from T. fusca in complex with a non-hydrolysable substrate analogue has been determined to 1.5 Å resolution. The glycosyl unit in subsite –1 was sterically hindered by Tyr73 and forced into a distorted 2SO conformation. In the enzyme where Tyr73 was mutated to a serine residue the hindrance was removed and the glycosyl unit in subsite –1 had a relaxed 4C1 chair conformation.