Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 65095
Title Comparative structural analysis and substrate specificity engineering of the hyperthermostable b-glucosidase CelB from Pyrococcus furiosus
Author(s) Kaper, T.; Lebbink, J.H.G.; Pouwels, J.; Kopp, J.; Schulz, G.E.; Oost, J. van der; Vos, W.M. de
Source Biochemistry 39 (2000). - ISSN 0006-2960 - p. 4963 - 4970.
DOI https://doi.org/10.1021/bi992463r
Department(s) Microbiology
VLAG
Publication type Refereed Article in a scientific journal
Publication year 2000
Abstract The substrate specificity of the -glucosidase (CelB) from the hyperthermophilic archaeon Pyrococcus furiosus, a family 1 glycosyl hydrolase, has been studied at a molecular level. Following crystallization and X-ray diffraction of this enzyme, a 3.3 Å resolution structural model has been obtained by molecular replacement. CelB shows a homo-tetramer configuration, with subunits having a typical ()8-barrel fold. Its active site has been compared to the one of the previously determined 6-phospho--glycosidase (LacG) from the mesophilic bacterium Lactococcus lactis. The overall design of the substrate binding pocket is very well conserved, with the exception of three residues that have been identified as a phosphate binding site in LacG. To verify the structural model and alter its substrate specificity, these three residues have been introduced at the corresponding positions in CelB (E417S, M424K, F426Y) in different combinations: single, double, and triple mutants. Characterization of the purified mutant CelB enzyme revealed that F426Y resulted in an increased affinity for galactosides, whereas M424K gave rise to a shifted pH optimum (from 5.0 to 6.0). Analysis of E417S revealed a 5-fold and a 3-fold increase of the efficiency of hydrolyzing o-nitrophenol--D-galactopyranoside-6-phosphate, in the single and triple mutants, respectively. In contrast, their activity on nonphosphorylated sugars was largely reduced (30-300-fold). The residue at position E417 in CelB seems to be the determining factor for the difference in substrate specificity between the two types of family 1 glycosidases.
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