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Structural Studies of Three GlycosidasesLarsson, Anna January 2006 (has links)
<p>Glycosidases hydrolyse the glycosidic bond in carbohydrates. Structural studies of three glycosidases with different substrate specificities are presented in this work.</p><p>Dextranase catalyzes the hydrolysis of <i>α</i>-1,6-glycosidic linkage in dextran polymers. The structure of dextranase, Dex49A, from <i>Penicillium minioluteum</i> 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.</p><p>Endo-<i>β</i>-1,4-D-mannanase catalyzes the depolymerization of <i>β</i>-1,4-mannan polymers. The structure of endo-1,4-<i>β</i>-mannanase Man5A from blue mussel <i>Mytilus edulis</i> 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. </p><p>Both the Dex49A and the Man5A structures were determined by multiple-wavelength anomalous diffraction using the selenium <i>K</i>-edge with selenomethionyl enzymes expressed in the yeast <i>Pichia pastoris</i>.</p><p>Endoglucanase Cel6A from <i>Thermobifida fusca</i> hydrolyzes the <i>β</i>-1,4 linkages in cellulose. The structure of the catalytic domain of Cel6A from <i>T. fusca</i> 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 <sup>2</sup>S<sub>O</sub> 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 <sup>4</sup>C<sub>1</sub> chair conformation.</p>
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Structural Studies of Three GlycosidasesLarsson, Anna January 2006 (has links)
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.
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