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Comparative structural analysis and substrate specificity engineering of the hyperthermostable beta-glucosidase CelB
T Kaper1, J H Lebbink, J Pouwels
1Laboratory of Microbiology, Department of Biomolecular Sciences, Wageningen University, Hesselink van Suchtelenweg 4, NL-6703 CT Wageningen, The Netherlands. thijs.kaper@algemeen.micr.wau.nl
Biochemistry
|May 23, 2000
Summary
Researchers studied beta-glucosidase (CelB) from Pyrococcus furiosus, a hyperthermophilic archaeon. Mutating specific residues altered its substrate specificity, revealing key factors in enzyme function.
Area of Science:
- Biochemistry
- Structural Biology
- Enzymology
Background:
- Beta-glucosidase (CelB) from the hyperthermophilic archaeon Pyrococcus furiosus is a family 1 glycosyl hydrolase.
- Understanding enzyme substrate specificity is crucial for biocatalysis and drug development.
Purpose of the Study:
- To investigate the molecular basis of substrate specificity in CelB.
- To engineer CelB for altered substrate preferences by introducing mutations based on structural comparisons.
Main Methods:
- Crystallization and X-ray diffraction to obtain a 3.3 A resolution structural model of CelB.
- Site-directed mutagenesis to introduce specific residue changes (E417S, M424K, F426Y).
- Biochemical characterization of purified wild-type and mutant CelB enzymes.
Main Results:
- The crystal structure revealed a conserved substrate binding pocket compared to a mesophilic homolog (LacG).
- Mutations F426Y increased galactoside affinity; M424K shifted the pH optimum.
- The E417S mutation significantly enhanced activity on phosphorylated substrates while reducing activity on non-phosphorylated sugars.
Conclusions:
- The residue at position 417 (E417) in CelB is a key determinant for substrate specificity differences between related glycosidases.
- Structural insights enable targeted engineering of enzyme function for specific applications.