Semi-rational approach for converting a GH36 α-glycosidase into an α-transglycosidase

Abstract

A large number of retaining glycosidases catalyze both hydrolysis and transglycosylation reactions. In order to use them as catalysts for oligosaccharide synthesis, the balance between these two competing reactions has to be shifted toward transglycosylation. We previously designed a semi-rational approach to convert the Thermus thermophilus β-glycosidases into transglycosidases by mutating highly conserved residues located around the -1 subsite. In an attempt to verify that this strategy could be a generic approach to turn glycosidases into transglycosidases, Geobacillus stearothermophilus α-galactosidase (AgaB) was selected in order to obtain α-transgalactosidases. This is of particular interest as, to date, there are no efficient α-galactosynthases, despite the considerable importance of α-galactooligosaccharides. Thus, by site-directed mutagenesis on 14 AgaB residues, 26 single mutants and 22 double mutants were created and screened, of which 11 single mutants and 6 double mutants exhibited improved synthetic activity, producing 4-nitrophenyl α-d-galactopyranosyl-(1,6)-α-d-galactopyranoside in 26-57% yields against only 22% when native AgaB was used. It is interesting to note that the best variant was obtained by mutating a second-shell residue, with no direct interaction with the substrate or a catalytic amino acid. As this approach has proved to be efficient with both α- and β-glycosidases, it is a promising route to convert retaining glycosidases into transglycosidases.

Rational Enzyme Design Without Structural Knowledge: A Sequence-Based Approach for Efficient Generation of Glycosylation Catalysts

“…However, such approaches require either in-depth structural and biochemical knowledge and, in some cases, use of sophisticated computational methods (rational design); or the creation of large libraries, introducing location-agnostic modifications, coupled with powerful phenotypic screens (random mutagenesis) 18 . Nevertheless, by simply targeting a small number of conserved active-site residues in several retaining GHs [19][20][21] , we previously demonstrated that transglycosylation capability can be improved without extensive screening using structural information coupled with sequence conservation analysis. Moreover, others have transposed our obtained beneficial mutations to related enzymes, leading to significant transglycosylation yields 22 .…”

A Single Point Mutation Alters the Transglycosylation/Hydrolysis Partition, Significantly Enhancing the Synthetic Capability of an endo-Glycoceramidase

“…Based on previous studies aimed at improving transglycosylation in rGHs, 14,16,17,[50][51][52][53][54] it is well-established that mutations that lower the hydrolytic component of the reaction and others that increase acceptor binding can lead to overall improvements in transglycosylation.…”

“…Interestingly, the key molecular determinants that differentiate TGs from hydrolytic GHs are still unclearly defined, despite considerable efforts to discover these and relevant studies on reversed transglycosylation/hydrolysis (T/H) partition. [12][13][14][15][16][17] The class of GHs (retaining GHs, hereafter referred to as rGHs) mostly holding the potential to perform transglycosylation reactions are those that catalyze the hydrolysis of glycosidic bonds through a double displacement mechanism that results in the retention (in the product) of the configuration of the anomeric carbon originally present in the donor substrate. The reaction trajectory of rGHs is characterized by the formation of a more or less transient glycosylated enzyme reaction intermediate that can be deglycosylated either by a water molecule (hydrolysis), or by another suitable acceptor moiety (transglycosylation), including sugars and alcohols ( Figure 1).…”

β-N-Acetylhexosaminidases for Carbohydrate Synthesis via Trans-Glycosylation

“…Another generic approach to increase trans-glycosylation activity of CAZymes is the mutation of other conserved residues in the active site to structurally related residues (e.g., from Tyr to Phe), which was first described in 2014 for a GH1 β-glycosidase [143] and has been transferred to many other GH families since then [144][145][146]. Recently, six conserved positions were identified from an alignment of 585 GH20 sequences and their effect on the trans-glycosylation activity of BbhI was studied [102].…”