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
85(5).
p.872-884
- 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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/6f8d81f6-0f1b-4c88-b826-74a90d2505b6
- author
- 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
- organization
- publishing date
- 2017-05
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Proteins: Structure, Function and Bioinformatics
- volume
- 85
- issue
- 5
- pages
- 872 - 884
- publisher
- John Wiley & Sons Inc.
- external identifiers
-
- pmid:28142197
- 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
- 2022-04-24 22:32:18
@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}},
number = {{5}},
pages = {{872--884}},
publisher = {{John Wiley & Sons Inc.}},
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}},
doi = {{10.1002/prot.25256}},
volume = {{85}},
year = {{2017}},
}