title: Rational design of a thermostable glycoside hydrolase from family 3 introduces β-glycosynthase activity.
abstract: The thermostable β-glucosidase from Thermotoga neapolitana, TnBgl3B, is a monomeric three-domain 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 KMThis 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%.
authors: Department of Chemistry, Biotechnology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden tania.pozzo@gmail.com.
authors: Tania
authors: T
authors: Pozzo
authors: Laboratory of Biochemistry, Institut Quimíc de Sarrià, Universitat Ramon Llull, Via Augusta 390, E-08017 Barcelona, Spain.
authors: Javier
authors: J
authors: Romero-García
authors: Laboratory of Biochemistry, Institut Quimíc de Sarrià, Universitat Ramon Llull, Via Augusta 390, E-08017 Barcelona, Spain.
authors: Magda
authors: M
authors: Faijes
authors: Laboratory of Biochemistry, Institut Quimíc de Sarrià, Universitat Ramon Llull, Via Augusta 390, E-08017 Barcelona, Spain.
authors: Antoni
authors: A
authors: Planas
authors: Department of Chemistry, Biotechnology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden.
authors: Eva
authors: E
authors: Nordberg Karlsson
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004795:Enzyme Stability / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods*; D008958:Models, Molecular; D009154:Mutation; D013379:Substrate Specificity; D046949:Thermotoga neapolitana / Q000201:enzymology*; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics
chemical_list: D006026:Glycoside Hydrolases; D001617:beta-Glucosidase
keywords: Thermotoga neapolitana; formate; quercetin; α-glucosyl fluoride; β-glycosynthase