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Preparation and crystallization of selenomethionyl dextranase from Penicillium minioluteum expressed in Pichia pastoris.
Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
2002 In: Acta Crystallogr D Biol Crystallogr., ISSN 0907-4449, Vol. 58, no 2, 346-348 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2002. Vol. 58, no 2, 346-348 p.
Identifiers
URN: urn:nbn:se:uu:diva-94011OAI: oai:DiVA.org:uu-94011DiVA: diva2:167693
Available from: 2006-02-17 Created: 2006-02-17Bibliographically approved
In thesis
1. Structural Studies of Three Glycosidases
Open this publication in new window or tab >>Structural Studies of Three Glycosidases
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 47 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 145
Keyword
Cell and molecular biology, glycosidase, dextranase, mannanase, endoglucanase, SeMet, MAD, crystal structure, Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-6339 (URN)91-554-6461-0 (ISBN)
Public defence
2006-03-10, B22, Biomedical centre, Husargatan 3, Uppsala, 10:00
Opponent
Supervisors
Available from: 2006-02-17 Created: 2006-02-17Bibliographically approved

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