Comparative structural analysis and substrate specificity engineering of the hyperthermostable beta-glucosidase CelB from Pyrococcus furiosus.
The substrate specificity of the beta-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 A resolution structural model has been obtained by molecular replacement. CelB shows a homo-tetramer configuration, with subunits having a typical (betaalpha)(8)-barrel fold. Its active site has been compared to the one of the previously determined 6-phospho-beta-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-beta-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.
Kaper
T
T
Laboratory of Microbiology, Department of Biomolecular Sciences, Wageningen University, Hesselink van Suchtelenweg 4, NL-6703 CT Wageningen, The Netherlands. thijs.kaper@algemeen.micr.wau.nl
Lebbink
J H
JH
Pouwels
J
J
Kopp
J
J
Schulz
G E
GE
van der Oost
J
J
de Vos
W M
WM
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015202:Protein Engineering; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D019843:Archaeal Proteins; D001426:Bacterial Proteins; C406923:CelB protein, Pyrococcus furiosus; D001617:beta-Glucosidase