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Three-dimensional crystal structure and enzymic characterization of beta-mannanase Man5A from blue mussel Mytilus edulis
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Physical and Analytical Chemistry, Surface Biotechnology.
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2006 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 14, no 357, 1500-1510 p.Article in journal (Refereed) Published
Abstract [en]

Endo-beta-1,4-d-mannanase is the key depolymerizing enzyme for beta-1,4-mannan polymers present in the cell walls of plants and some algae, as well as in some types of plant seeds. Endo-1,4-beta-mannanase from blue mussel Mytilus edulis (MeMan5A) belongs to the glycoside hydrolase (GH) family 5 enzymes. The MeMan5A structure has been determined to 1.6A resolution using the multiple-wavelength anomalous dispersion method at the selenium K edge with selenomethionyl MeMan5A expressed in the yeast Pichia pastoris. As expected for GH 5 enzymes, the structure showed a (betaalpha)(8)-barrel fold. An unusually large number of histidine side-chains are exposed on the surface, which may relate to its location within the crystalline style of the digestive tract of the mussel. Kinetic analysis of MeMan5A revealed that the enzyme requires at least six subsites for efficient hydrolysis. Mannotetraose (M4) and mannopentaose (M5) were shown to interact with subsites -3 to +1, and -3 to +2, respectively. A clear kinetic threshold was observed when going from M4 to M5, indicating that the +2 subsite provides important interaction in the hydrolysis of short oligomeric mannose substrates. The catalytic centre motif at subsite -1 found in superfamily GH clan A is, as expected, conserved in MeMan5A, but the architecture of the catalytic cleft differs significantly from other GH 5 enzyme structures. We therefore suggest that MeMan5A represents a new subfamily in GH 5.

Place, publisher, year, edition, pages
2006. Vol. 14, no 357, 1500-1510 p.
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:uu:diva-94014DOI: 10.1016/j.jmb.2006.01.044PubMedID: 16487541OAI: oai:DiVA.org:uu-94014DiVA: diva2:167696
Available from: 2006-02-17 Created: 2006-02-17 Last updated: 2013-07-10Bibliographically 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
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Available from: 2006-02-17 Created: 2006-02-17Bibliographically approved

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Janson, Jan-Christer

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