Semi-rational approach for converting a GH1  -glycosidase into a  -transglycosidase

Abstract

A large number of retaining glycosidases catalyze both hydrolysis and transglycosylation reactions, but little is known about what determines the balance between these two activities (transglycosylation/hydrolysis ratio). We previously obtained by directed evolution the mutants F401S and N282T of Thermus thermophilus β-glycosidase (Ttβ-gly, glycoside hydrolase family 1 (GH1)), which display a higher transglycosylation/hydrolysis ratio than the wild-type enzyme. In order to find the cause of these activity modifications, and thereby set up a generic method for easily obtaining transglycosidases from glycosidases, we determined their X-ray structure. No major structural changes could be observed which could help to rationalize the mutagenesis of glycosidases into transglycosidases. However, as these mutations are highly conserved in GH1 β-glycosidases and are located around the -1 site, we pursued the isolation of new transglycosidases by targeting highly conserved amino acids located around the active site. Thus, by single-point mutagenesis on Ttβ-gly, we created four new mutants that exhibit improved synthetic activity, producing disaccharides in yields of 68-90% against only 36% when native Ttβ-gly was used. As all of the chosen positions were well conserved among GH1 enzymes, this approach is most probably a general route to convert GH1 glycosidases into transglycosidases.

Comparison of lipases and glycoside hydrolases as catalysts in synthesis reactions

“…A β-glycosidase from Thermus thermophilus modified in this way produced 60 and 75% transglycosylation yield from maltose and cellobiose, respectively, compared to 6 and 8% for the wild-type enzyme (Feng et al 2005). X-ray structures of wild-type and mutants with improved selectivity for transglycosylation vs. hydrolysis were compared, and no major structural changes were observed (Teze et al 2014). It was noted that several mutations having a positive effect on the observed transglycosylation/hydrolysis ratio occurred in highly conserved residues in the −1 binding site (Teze et al 2014).…”

“…X-ray structures of wild-type and mutants with improved selectivity for transglycosylation vs. hydrolysis were compared, and no major structural changes were observed (Teze et al 2014). It was noted that several mutations having a positive effect on the observed transglycosylation/hydrolysis ratio occurred in highly conserved residues in the −1 binding site (Teze et al 2014). Mutating such residues, in T. thermophilus β-glycosidase, produced four new mutants with increased selectivity for transglycosylation.…”

Active site cleft mutants of Os9BGlu31 transglucosidase modify acceptor substrate specificity and allow production of multiple kaempferol glycosides

“…There are a 57 large number of retaining GHs that catalyze both hydrolysis and 58 transglycosylations, but not much is known regarding what determines 59 the ratio between the two types of reactions [6]. 60 Glycosynthases, which transfer sugars from anomeric fluoroglu-61 cosides, are typically produced by mutation of a retaining GH cata-62 lytic nucleophile to a small non-nucleophilic group [7,8], while 63 thioglycoligases, which transfer from an activated donor to a thiol (or 64 other nucleophilic) acceptor, are typically made by mutation of the 65 catalytic acid/base [9].…”

“…These mutational approaches modify the mecha-66 nism to disfavor hydrolysis and therefore improve transglycosylation 67 yields. Other groups have worked to convert GH to TG [6] or enhance 68 the rate and substrate specificity of natural transglycosidases, 69 such as amylosucrases and cyclodextrinases, by directed evolution 70 [10]. In GH family GH16, a surface loop thought to affect acceptor 71 substrate binding was identified to help differentiate between a 72 xyloglucan endohydrolase and xyloglucan endotransferase (XET, a 73 TG) [11], but the critical determinant of transglycosylation activity 74 is generally unclear.…”

“…The UPLC-MSMS chromatograms of the deduced products in negative ion mode are shown with the provisionally identified products labeled with numbers as follows: kaempferol 3,7-bis-O-glucoside (1); kaempferol 4′,7-bis-O-glucoside (2); kaempferol 3,4′-bis-O-glucoside (3); kaempferol 3-O-glucoside and 4NP (4); kaempferol 7-O-glucoside (5); kaempferol 4′-O-glucoside(6); and kaempferol(7).…”