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Tailoring the substrate specificity of the β‐glycosidase from the thermophilic archaeon Sulfolobus solfataricus
Author(s) -
Corbett Kevin,
Fordham-Skelton Anthony P.,
Gatehouse John A.,
Davis Benjamin G.
Publication year - 2001
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(01)03154-4
Subject(s) - sulfolobus solfataricus , glycoside hydrolase , thermophile , biochemistry , sulfolobus , chemistry , galactose , fucose , thermostability , escherichia coli , mutant , directed evolution , stereochemistry , enzyme , archaea , gene
The substrate specificity of the thermophilic β‐glycosidase ( lacS ) from the archaeon Sulfolobus solfataricus (SSβG), 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 SSβG mutants in parallel synthesis were demonstrated by the formation of libraries of β‐glycosides of Glc, Gal, xylose, Man in one‐pot preparations at 50°C in the presence of organic solvents, that could not be performed by SSβG‐WT.