Rational design of a thermostable glycoside hydrolase from family 3 introduces β-glycosynthase activity

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Rational design of a thermostable glycoside hydrolase from family 3 introduces β-glycosynthase activity. / Pozzo, Tania; Romero-García, Javier; Faijes, Magda; Planas, Antoni; Nordberg Karlsson, Eva.

In: Glycobiology, Vol. 27, No. 2, 2017, p. 165-175.

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Pozzo, Tania ; Romero-García, Javier ; Faijes, Magda ; Planas, Antoni ; Nordberg Karlsson, Eva. / Rational design of a thermostable glycoside hydrolase from family 3 introduces β-glycosynthase activity. In: Glycobiology. 2017 ; Vol. 27, No. 2. pp. 165-175.

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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 - Nordberg Karlsson, Eva

N1 - © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

PY - 2017

Y1 - 2017

N2 - 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 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 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 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%.

U2 - 10.1093/glycob/cww081

DO - 10.1093/glycob/cww081

M3 - Article

VL - 27

SP - 165

EP - 175

JO - Glycobiology

T2 - Glycobiology

JF - Glycobiology

SN - 1460-2423

IS - 2

ER -