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5IDI

Structure of beta glucosidase 1A from Thermotoga neapolitana, mutant E349A

Summary for 5IDI
Entry DOI10.2210/pdb5idi/pdb
Descriptor1,4-beta-D-glucan glucohydrolase, ACETATE ION (3 entities in total)
Functional Keywordsbeta-glucosidase, glycosyl hydrolase family 1, hydrolase
Biological sourceThermotoga neapolitana
Total number of polymer chains2
Total formula weight105403.37
Authors
Kulkarni, T.,Nordberg Karlsson, E.,Logan, D.T. (deposition date: 2016-02-24, release date: 2017-02-08, Last modification date: 2024-01-10)
Primary citationKulkarni, T.S.,Khan, S.,Villagomez, R.,Mahmood, T.,Lindahl, S.,Logan, D.T.,Linares-Pasten, J.A.,Nordberg Karlsson, E.
Crystal structure of beta-glucosidase 1A from Thermotoga neapolitana and comparison of active site mutants for hydrolysis of flavonoid glucosides.
Proteins, 85:872-884, 2017
Cited by
PubMed 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. Proteins 2017; 85:872-884. © 2016 Wiley Periodicals, Inc.
PubMed: 28142197
DOI: 10.1002/prot.25256
PDB entries with the same primary citation
Experimental method
X-RAY DIFFRACTION (1.9 Å)
Structure validation

226707

數據於2024-10-30公開中

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