26255-70-9Relevant articles and documents
Rational design of a thermostable glycoside hydrolase from family 3 introduces β-glycosynthase activity
Pozzo, Tania,Romero-García, Javier,Faijes, Magda,Planas, Antoni,Karlsson, Eva Nordberg
, p. 165 - 175 (2017)
The thermostable β-glucosidase from Thermotoga neapolitana, TnBgl3B, is a monomeric threedomain representative from glycoside hydrolase family 3. By using chemical reactivation with exogenous nucleophiles in previous studies with TnBg13B, the catalytic nucleophile (D242) and corresponding acid/base residue (E458) were determined. Identifying these residues led to the attempt of converting TnBgl3B into a β-glucosynthase, where three nucleophilic variants were created (TnBgl3B-D242G, TnBgl3B-D242A, TnBgl3B-D242S) and all of them failed to exhibit glucosynthase activity. A deeper analysis of the TnBgl3B active site led to the generation of three additional variants, each of which received a single-point mutation. Two of these variants were altered at the -1 subsite (Y210F, W243F) and the third received a substitution near the binding site's aglycone region (N248R). Kinetic evaluation of these three variants revealed that W243F substitution reduced hydrolytic turnover while maintaining KM. This key W243F mutation was then introduced into the original nucleophile variants and the resulting double mutants were successfully converted into β-glucosynthases that were assayed using two separate biosynthetic methods. The first reaction used an α-glucosyl fluoride donor with a 4-nitrophenyl-β-D-glucopyranoside (4NPGlc) acceptor, and the second used 4NPGlc as both the donor and acceptor in the presence of the exogenous nucleophile formate. The primary specificity observed was a β-1,3-linked disaccharide product, while a secondary β-1,4-linked disaccharide product was observed with increased incubation times. Additional analysis revealed that substituting quercetin-3-glycoside for the second reaction's acceptor molecule resulted in the successful production of quercetin-3,4'-diglycosides with yields up to 40%.
Glycosynthases from Thermotoga neapolitana β-glucosidase 1A: A comparison of α-glucosyl fluoride and in situ-generated α-glycosyl formate donors
Pozzo, Tania,Plaza, Merichel,Romero-Garcia, Javier,Faijes, Magda,Karlsson, Eva Nordberg,Planas, Antoni
, p. 132 - 139 (2014/07/21)
TnBgl1A from the thermophile Thermotoga neapolitana is a dimeric β-glucosidase that belongs to glycoside hydrolase family 1 (GH1), with hydrolytic activity through the retaining mechanism, and a broad substrate specificity acting on β-1,4-, β-1,3- and β-1,6-linkages over a range of glyco-oligosaccharides. Three variants of the enzyme (TnBgl1A-E349G, TnBgl1A-E349A and TnBgl1A-E349S), mutated at the catalytic nucleophile, were constructed to evaluate their glycosynthase activity towards oligosaccharide synthesis. Two approaches were used for the synthesis reactions, both of which utilized 4-nitrophenyl β-d-glucopyranoside (4NPGlc) as an acceptor molecule: the first using an α-glucosyl fluoride donor at low temperature (35 °C) in a classical glycosynthase reaction, and the second by in situ generation of the glycosyl donor with (4NPGlc), where formate served as the exogenous nucleophile under higher temperature (70 °C). Using the first approach, TnBgl1A-E349G and TnBgl1A-E349A synthesized disaccharides with β-1,3-linkages in good yields (up to 61%) after long incubations (15 h). However, the GH1 glycosynthase Bgl3-E383A from a mesophilic Streptomyces sp., used as reference enzyme, generated a higher yield at the same temperature with both a shorter reaction time and a lower enzyme concentration. The second approach yielded disaccharides for all three mutants with predominantly β-1,3-linkages (up to 45%) but also β-1,4-linkages (up to 12.5%), after 7 h reaction time. The TnBgl1A glycosynthases were also used for glycosylation of flavonoids, using the two described approaches. Quercetin-3-glycoside was tested as an acceptor molecule and the resultant product was quercetin-3,4′-diglycosides in significantly lower yields, indicating that TnBgl1A preferentially selects 4NPGlc as the acceptor.
Creation of an α-mannosynthase from a broad glycosidase scaffold
Yamamoto, Keisuke,Davis, Benjamin G.
supporting information; experimental part, p. 7449 - 7453 (2012/09/21)
α-Mannosides made easy: Mutation of a family-GH31 α-glucosidase that displays plasticity to alterations at the 2-OH position of donor substrates created an efficient α-mannoside-synthesizing biocatalyst. A simple fluoride donor reagent was used for the synthesis of a range of mono-α-mannosylated conjugates using the α-mannosynthase displaying low (unwanted) oligomerization activity. Copyright
