Tailoring the substrate specificity of the beta-glycosidase from the thermophilic archaeon Sulfolobus solfataricus.
The substrate specificity of the thermophilic beta-glycosidase (lacS) from the archaeon Sulfolobus solfataricus (SSbetaG), a member of the glycohydrolase family 1, has been analysed at a molecular level using predictions from known protein sequences and structures and through site-directed mutagenesis. Three critical residues were identified and mutated to create catalysts with altered and broadened specificities for use in glycoside synthesis. The wild-type (WT) and mutated sequences were expressed as recombinant fusion proteins in Escherichia coli, with an added His(6)-tag to allow one-step chromatographic purification. Consistent with side-chain orientation towards OH-6, the single Met439-->Cys mutation enhances D-xylosidase specificity 4.7-fold and decreases D-fucosidase activity 2-fold without greatly altering its activity towards other D-glycoside substrates. Glu432-->Cys and Trp433-->Cys mutations directed towards OH-4 and -3, respectively, more dramatically impair glucose (Glc), galactose (Gal), fucose specificity than for other glycosides, resulting in two glycosidases with greatly broadened substrate specificities. These include the first examples of stereospecificity tailoring in glycosidases (e.g. WT-->W433C, k(cat)/K(M) (Gal):k(cat)/K(M) (mannose (Man))=29.4:1-->1.2:1). The robustness and high utility of these broad specificity SSbetaG mutants in parallel synthesis were demonstrated by the formation of libraries of beta-glycosides of Glc, Gal, xylose, Man in one-pot preparations at 50 degrees C in the presence of organic solvents, that could not be performed by SSbetaG-WT.
Corbett
K
K
Department of Chemistry, University of Durham, UK.
Fordham-Skelton
A P
AP
Gatehouse
J A
JA
Davis
B G
BG
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D005959:Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008715:Methionine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology*
D018698:Glutamic Acid; D008715:Methionine; D005959:Glucosidases; C104851:Sulfolobus solfataricus beta-glycosidase