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Crystal structure of β-glucosidase 1A from Thermotoga neapolitana and comparison of active site mutants for hydrolysis of flavonoid glucosides

Kulkarni, Tejas LU ; Khan, Samiullah LU ; Villagomez, Rodrigo LU ; Mahmood, Tahir; Lindahl, Sofia LU ; Logan, Derek T. LU ; Linares-Pastén, Javier LU and Nordberg Karlsson, Eva LU (2017) In Proteins: Structure, Function and Bioinformatics
Abstract
The β-glucosidase TnBgl1A catalyses hydrolysis of O-linked terminal β-glycosidic bonds at the nonreducing end of glycosides/oligosaccharides. Enzymes with this specificity have potential in lignocellulose conversion (degrading cellobiose to glucose) and conversion of bioactive flavonoids (modification of glycosylation results in modulation of bioavailability). Previous work has shown TnBgl1A to hydrolyse 3, 4′ and 7 glucosylation in flavonoids, and although conversion of 3-glucosylated substrate to aglycone was low, it was improved by mutagenesis of residue N220. To further explore structure-function relationships, the crystal structure of the nucleophile mutant TnBgl1A-E349G was determined at 1.9 Å resolution, and docking studies of... (More)
The β-glucosidase TnBgl1A catalyses hydrolysis of O-linked terminal β-glycosidic bonds at the nonreducing end of glycosides/oligosaccharides. Enzymes with this specificity have potential in lignocellulose conversion (degrading cellobiose to glucose) and conversion of bioactive flavonoids (modification of glycosylation results in modulation of bioavailability). Previous work has shown TnBgl1A to hydrolyse 3, 4′ and 7 glucosylation in flavonoids, and although conversion of 3-glucosylated substrate to aglycone was low, it was improved by mutagenesis of residue N220. To further explore structure-function relationships, the crystal structure of the nucleophile mutant TnBgl1A-E349G was determined at 1.9 Å resolution, and docking studies of flavonoid substrates were made to reveal substrate interacting residues. A series of single amino acid changes were introduced in the aglycone binding region [N220(S/F), N221(S/F), F224(I), F310(L/E), and W322(A)] of the wild type. Activity screening was made on eight glucosylated flavonoids, and kinetic parameters were monitored for the flavonoid quercetin-3-glucoside (Q3), as well as for the model substrate para-nitrophenyl-β-d-glucopyranoside (pNPGlc). Substitution by Ser at N220 or N221 increased the catalytic efficiency on both pNPGlc and Q3. Residue W322 was proven important for substrate accomodation, as mutagenesis to W322A resulted in a large reduction of hydrolytic activity on 3-glucosylated flavonoids. Flavonoid glucoside hydrolysis was unaffected by mutations at positions 224 and 310. The mutations did not significantly affect thermal stability, and the variants kept an apparent unfolding temperature of 101°C. This work pinpoints positions in the aglycone region of TnBgl1A of importance for specificity on flavonoid-3-glucosides, improving the molecular understanding of activity in GH1 enzymes. (Less)
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organization
publishing date
type
Contribution to journal
publication status
epub
subject
in
Proteins: Structure, Function and Bioinformatics
publisher
John Wiley & Sons
external identifiers
  • scopus:85014685313
  • wos:000399380400007
ISSN
1097-0134
DOI
10.1002/prot.25256
language
English
LU publication?
yes
id
6f8d81f6-0f1b-4c88-b826-74a90d2505b6
date added to LUP
2017-03-09 21:25:10
date last changed
2017-09-18 11:38:29
@article{6f8d81f6-0f1b-4c88-b826-74a90d2505b6,
  abstract     = {The β-glucosidase TnBgl1A catalyses hydrolysis of O-linked terminal β-glycosidic bonds at the nonreducing end of glycosides/oligosaccharides. Enzymes with this specificity have potential in lignocellulose conversion (degrading cellobiose to glucose) and conversion of bioactive flavonoids (modification of glycosylation results in modulation of bioavailability). Previous work has shown TnBgl1A to hydrolyse 3, 4′ and 7 glucosylation in flavonoids, and although conversion of 3-glucosylated substrate to aglycone was low, it was improved by mutagenesis of residue N220. To further explore structure-function relationships, the crystal structure of the nucleophile mutant TnBgl1A-E349G was determined at 1.9 Å resolution, and docking studies of flavonoid substrates were made to reveal substrate interacting residues. A series of single amino acid changes were introduced in the aglycone binding region [N220(S/F), N221(S/F), F224(I), F310(L/E), and W322(A)] of the wild type. Activity screening was made on eight glucosylated flavonoids, and kinetic parameters were monitored for the flavonoid quercetin-3-glucoside (Q3), as well as for the model substrate para-nitrophenyl-β-d-glucopyranoside (pNPGlc). Substitution by Ser at N220 or N221 increased the catalytic efficiency on both pNPGlc and Q3. Residue W322 was proven important for substrate accomodation, as mutagenesis to W322A resulted in a large reduction of hydrolytic activity on 3-glucosylated flavonoids. Flavonoid glucoside hydrolysis was unaffected by mutations at positions 224 and 310. The mutations did not significantly affect thermal stability, and the variants kept an apparent unfolding temperature of 101°C. This work pinpoints positions in the aglycone region of TnBgl1A of importance for specificity on flavonoid-3-glucosides, improving the molecular understanding of activity in GH1 enzymes.},
  author       = {Kulkarni, Tejas and Khan, Samiullah and Villagomez, Rodrigo and Mahmood, Tahir and Lindahl, Sofia and Logan, Derek T. and Linares-Pastén, Javier and Nordberg Karlsson, Eva},
  issn         = {1097-0134},
  language     = {eng},
  month        = {03},
  publisher    = {John Wiley & Sons},
  series       = {Proteins: Structure, Function and Bioinformatics},
  title        = {Crystal structure of β-glucosidase 1A from Thermotoga neapolitana and comparison of active site mutants for hydrolysis of flavonoid glucosides},
  url          = {http://dx.doi.org/10.1002/prot.25256},
  year         = {2017},
}