Developing Promiscuous Glycosidases for Glycoside Synthesis: Residues W433 and E432 in Sulfolobus solfataricus β‐Glycosidase are Important Glucoside‐ and Galactoside‐Specificity Determinants

Abstract

Two residues that have been implicated in determining the substrate specificity of the thermophilic beta-glycosidase from the archaeon Sulfolobus solfataricus (SsbetaG), a member of the glycosyl hydrolase family 1, have been mutated by site-directed mutagenesis so as to create more versatile catalysts for carbohydrate chemistry. The wild-type and mutated sequences were expressed in E. coli with a His(7)-tag to allow one-step chromatographic purification. The E432C and W433C mutations removed key interactions with the OH-4 and OH-3 of the sugar substrates, thus reducing the discrimination of glucose, galactose and fucose with respect to other glycosides. This resulted in two glycosidases with greatly broadened substrate specificities. Observed changes include a 24-fold increase in Man:Gal activity and an 18-fold increase in GalA:Gal activity. This promiscuous substrate tolerance was further illustrated by the parallel synthesis of a beta-glycoside library of glucose, galactose, xylose and mannose in one pot at 50 degrees C, in organic solvent. The synthetic potential of the catalysts was further evaluated through alkyl glycoside transglycosylation yields, including the first examples of synthesis of beta-mannosides and beta-xylosides with SsbetaG.

Genome mining and motif modifications of glycoside hydrolase family 1 members encoded by Geobacillus kaustophilus HTA426 provide thermostable 6-phospho-β-glycosidase and β-fucosidase

“…GH1 members also catalyze the formation of glycosidic linkages by thermodynamically controlled reverse reactions and/or kinetically controlled transglycosylations (Corbett et al 2001;Hancock et al 2005;Trincone et al 1991). These properties indicate the potential of GH1 members for use as saccharolytic catalysts and as robust, simple, and inexpensive glycosylation catalysts for the synthesizing glycoconjugates, such as therapeutic oligosaccharides (Benešová et al 2010;Corbett et al 2001;Hancock et al 2005).…”

“…The catalytic activity of GH1 members involves a double displacement mechanism through the formation of a covalent glycosylenzyme intermediate, in which two glutamate residues are involved; one acts as a general acid/base and the other acts as a nucleophile (Kempton and Withers 1992;Zechel and Withers 2000). GH1 members also catalyze the formation of glycosidic linkages by thermodynamically controlled reverse reactions and/or kinetically controlled transglycosylations (Corbett et al 2001;Hancock et al 2005;Trincone et al 1991). These properties indicate the potential of GH1 members for use as saccharolytic catalysts and as robust, simple, and inexpensive glycosylation catalysts for the synthesizing glycoconjugates, such as therapeutic oligosaccharides (Benešová et al 2010;Corbett et al 2001;Hancock et al 2005).…”

Site-Directed Mutagenesis as Applied to Biocatalysts

“…For example, rational modification of the -glycosidase from Sulfolobus sulfataricus to accept a wider range of substrates in transglycosylation reactions has been done. Site-directed mutagenesis was used to alter two key residues involved in substrate recognition to provide access to many different glycoside linkages, including the especially problematic -mannosyl and -xylosyl linkages (Hancock et al, 2005). We will focus our discussion on the protein engineering work on -amylases carried out by us and others.…”

Characterization of Five β-Glycoside Hydrolases from Cellulomonas fimi ATCC 484

“…The family contains various activities such as exoacting ␤-D-glucosidases, ␣-L-arabinofuranosidases, ␤-D-xylopyranosidases, and N-acetyl-␤-D-glucosaminidases, all of which use a retaining glycosidase mechanism (7)(8)(9). In addition to hydrolytic activities, some GH3 enzymes may catalyze glycosidic bond formation via either a thermodynamically controlled reverse hydrolysis or a kinetically controlled transglycosylation (10,11). Together, these enzymes are responsible for cellulosic biomass degradation, plant and bacterial cell wall remodeling, energy metabolism, and defense against pathogens (7,12).…”