TY - JOUR
T1 - Rational design of a thermostable glycoside hydrolase from family 3 introduces β-glycosynthase activity
AU - Pozzo, Tania
AU - Romero-García, Javier
AU - Faijes, Magda
AU - Planas, Antoni
AU - Karlsson, Eva Nordberg
N1 - Publisher Copyright:
© The Author 2016.
PY - 2017
Y1 - 2017
N2 - 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%.
AB - 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%.
KW - Formate
KW - Quercetin
KW - Thermotoga neapolitana
KW - α-glucosyl fluoride
KW - β-glycosynthase
UR - http://www.scopus.com/inward/record.url?scp=85014428006&partnerID=8YFLogxK
U2 - 10.1093/glycob/cww081
DO - 10.1093/glycob/cww081
M3 - Article
C2 - 27496759
AN - SCOPUS:85014428006
SN - 0959-6658
VL - 27
SP - 165
EP - 175
JO - Glycobiology
JF - Glycobiology
IS - 1
ER -