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Glycosynthases from Thermotoga neapolitana beta-glucosidase 1A: A comparison of alpha-glucosyl fluoride and in situ-generated alpha-glycosyl formate donors

Pozzo, Tania LU ; Plaza, Merichel LU ; Romero-Garcia, Javier; Faijes, Magda; Nordberg Karlsson, Eva LU and Planas, Antoni (2014) In Journal of Molecular Catalysis B: Enzymatic 107. p.132-139
Abstract
TnBgl1A from the thermophile Thermotoga neapolitana is a dimeric beta-glucosidase that belongs to glycoside hydrolase family 1 (GH1), with hydrolytic activity through the retaining mechanism, and a broad substrate specificity acting on beta-1,4-, beta-1,3- and beta-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 beta-D-glucopyranoside (4NPGIc) as an acceptor molecule: the first using an alpha-glucosyl fluoride donor at low temperature (35... (More)
TnBgl1A from the thermophile Thermotoga neapolitana is a dimeric beta-glucosidase that belongs to glycoside hydrolase family 1 (GH1), with hydrolytic activity through the retaining mechanism, and a broad substrate specificity acting on beta-1,4-, beta-1,3- and beta-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 beta-D-glucopyranoside (4NPGIc) as an acceptor molecule: the first using an alpha-glucosyl fluoride donor at low temperature (35 degrees C) in a classical glycosynthase reaction, and the second by in situ generation of the glycosyl donor with (4NPGIc), where formate served as the exogenous nucleophile under higher temperature (70 degrees C). Using the first approach, TnBgl1A_E349G and TnBgl1A_E349A synthesized disaccharides with beta-1,3-linkages in good yields (up to 61%) after long incubations (15 h). However, the GH1 glycosynthase Bg13_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 beta-1,3-linkages (up to 45%) but also beta-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 4NPGIc as the acceptor. (C) 2014 Elsevier B.V. All rights reserved. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
beta-Glucosidase, alpha-Glucosyl fluoride, Formate, 4-Nitrophenyl, beta-D-glucopyranoside, Quercetin
in
Journal of Molecular Catalysis B: Enzymatic
volume
107
pages
132 - 139
publisher
Elsevier
external identifiers
  • wos:000340698300019
  • scopus:84903720920
ISSN
1873-3158
DOI
10.1016/j.molcatb.2014.05.021
language
English
LU publication?
yes
id
019d420c-82c6-4133-ba98-712b190e5640 (old id 4648917)
date added to LUP
2014-09-24 08:55:09
date last changed
2017-10-01 03:22:04
@article{019d420c-82c6-4133-ba98-712b190e5640,
  abstract     = {TnBgl1A from the thermophile Thermotoga neapolitana is a dimeric beta-glucosidase that belongs to glycoside hydrolase family 1 (GH1), with hydrolytic activity through the retaining mechanism, and a broad substrate specificity acting on beta-1,4-, beta-1,3- and beta-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 beta-D-glucopyranoside (4NPGIc) as an acceptor molecule: the first using an alpha-glucosyl fluoride donor at low temperature (35 degrees C) in a classical glycosynthase reaction, and the second by in situ generation of the glycosyl donor with (4NPGIc), where formate served as the exogenous nucleophile under higher temperature (70 degrees C). Using the first approach, TnBgl1A_E349G and TnBgl1A_E349A synthesized disaccharides with beta-1,3-linkages in good yields (up to 61%) after long incubations (15 h). However, the GH1 glycosynthase Bg13_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 beta-1,3-linkages (up to 45%) but also beta-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 4NPGIc as the acceptor. (C) 2014 Elsevier B.V. All rights reserved.},
  author       = {Pozzo, Tania and Plaza, Merichel and Romero-Garcia, Javier and Faijes, Magda and Nordberg Karlsson, Eva and Planas, Antoni},
  issn         = {1873-3158},
  keyword      = {beta-Glucosidase,alpha-Glucosyl fluoride,Formate,4-Nitrophenyl,beta-D-glucopyranoside,Quercetin},
  language     = {eng},
  pages        = {132--139},
  publisher    = {Elsevier},
  series       = {Journal of Molecular Catalysis B: Enzymatic},
  title        = {Glycosynthases from Thermotoga neapolitana beta-glucosidase 1A: A comparison of alpha-glucosyl fluoride and in situ-generated alpha-glycosyl formate donors},
  url          = {http://dx.doi.org/10.1016/j.molcatb.2014.05.021},
  volume       = {107},
  year         = {2014},
}