Abstract
Rice BGlu1 β-glucosidase is an oligosaccharide exoglucosidase that binds to six β-(1→4)-linked glucosyl residues in its active site cleft. Here, we demonstrate that a BGlu1 E176Q active site mutant can be effectively rescued by small nucleophiles, such as acetate, azide and ascorbate, for hydrolysis of aryl glycosides in a pH-independent manner above pH. 5, consistent with the role of E176 as the catalytic acid-base. Cellotriose, cellotetraose, cellopentaose, cellohexaose and laminaribiose are not hydrolyzed by the mutant and instead exhibit competitive inhibition. The structures of the BGlu1 E176Q, its complexes with cellotetraose, cellopentaose and laminaribiose, and its covalent intermediate with 2-deoxy-2-fluoroglucoside were determined at 1.65, 1.95, 1.80, 2.80, and 1.90. Å resolution, respectively. The Q176. Nε was found to hydrogen bond to the glycosidic oxygen of the scissile bond, thereby explaining its high activity. The enzyme interacts with cellooligosaccharides through direct hydrogen bonds to the nonreducing terminal glucosyl residue. However, interaction with the other glucosyl residues is predominantly mediated through water molecules, with the exception of a direct hydrogen bond from N245 to glucosyl residue 3, consistent with the apparent high binding energy at this residue. Hydrophobic interactions with the aromatic sidechain of W358 appear to orient glucosyl residues 2 and 3, while Y341 orients glucosyl residues 4 and 5. In contrast, laminaribiose has its second glucosyl residue positioned to allow direct hydrogen bonding between its O2 and Q176 Oε and O1 and N245. These are the first GH1 glycoside hydrolase family structures to show oligosaccharide binding in the hydrolytic configuration.
| Original language | English |
|---|---|
| Pages (from-to) | 169-179 |
| Number of pages | 11 |
| Journal | Journal of Structural Biology |
| Volume | 173 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - Jan 1 2011 |
| Externally published | Yes |
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Keywords
- Acid-base mutant
- Cello-oligosaccharides
- Enzyme-substrate complex
- Glycoside hydrolase
- X-ray crystallography
ASJC Scopus subject areas
- Structural Biology
Cite this
The structural basis of oligosaccharide binding by rice BGlu1 beta-glucosidase. / Chuenchor, Watchalee; Pengthaisong, Salila; Robinson, Robert C.; Yuvaniyama, Jirundon; Svasti, Jisnuson; Cairns, James R.Ketudat.
In: Journal of Structural Biology, Vol. 173, No. 1, 01.01.2011, p. 169-179.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - The structural basis of oligosaccharide binding by rice BGlu1 beta-glucosidase
AU - Chuenchor, Watchalee
AU - Pengthaisong, Salila
AU - Robinson, Robert C.
AU - Yuvaniyama, Jirundon
AU - Svasti, Jisnuson
AU - Cairns, James R.Ketudat
PY - 2011/1/1
Y1 - 2011/1/1
N2 - Rice BGlu1 β-glucosidase is an oligosaccharide exoglucosidase that binds to six β-(1→4)-linked glucosyl residues in its active site cleft. Here, we demonstrate that a BGlu1 E176Q active site mutant can be effectively rescued by small nucleophiles, such as acetate, azide and ascorbate, for hydrolysis of aryl glycosides in a pH-independent manner above pH. 5, consistent with the role of E176 as the catalytic acid-base. Cellotriose, cellotetraose, cellopentaose, cellohexaose and laminaribiose are not hydrolyzed by the mutant and instead exhibit competitive inhibition. The structures of the BGlu1 E176Q, its complexes with cellotetraose, cellopentaose and laminaribiose, and its covalent intermediate with 2-deoxy-2-fluoroglucoside were determined at 1.65, 1.95, 1.80, 2.80, and 1.90. Å resolution, respectively. The Q176. Nε was found to hydrogen bond to the glycosidic oxygen of the scissile bond, thereby explaining its high activity. The enzyme interacts with cellooligosaccharides through direct hydrogen bonds to the nonreducing terminal glucosyl residue. However, interaction with the other glucosyl residues is predominantly mediated through water molecules, with the exception of a direct hydrogen bond from N245 to glucosyl residue 3, consistent with the apparent high binding energy at this residue. Hydrophobic interactions with the aromatic sidechain of W358 appear to orient glucosyl residues 2 and 3, while Y341 orients glucosyl residues 4 and 5. In contrast, laminaribiose has its second glucosyl residue positioned to allow direct hydrogen bonding between its O2 and Q176 Oε and O1 and N245. These are the first GH1 glycoside hydrolase family structures to show oligosaccharide binding in the hydrolytic configuration.
AB - Rice BGlu1 β-glucosidase is an oligosaccharide exoglucosidase that binds to six β-(1→4)-linked glucosyl residues in its active site cleft. Here, we demonstrate that a BGlu1 E176Q active site mutant can be effectively rescued by small nucleophiles, such as acetate, azide and ascorbate, for hydrolysis of aryl glycosides in a pH-independent manner above pH. 5, consistent with the role of E176 as the catalytic acid-base. Cellotriose, cellotetraose, cellopentaose, cellohexaose and laminaribiose are not hydrolyzed by the mutant and instead exhibit competitive inhibition. The structures of the BGlu1 E176Q, its complexes with cellotetraose, cellopentaose and laminaribiose, and its covalent intermediate with 2-deoxy-2-fluoroglucoside were determined at 1.65, 1.95, 1.80, 2.80, and 1.90. Å resolution, respectively. The Q176. Nε was found to hydrogen bond to the glycosidic oxygen of the scissile bond, thereby explaining its high activity. The enzyme interacts with cellooligosaccharides through direct hydrogen bonds to the nonreducing terminal glucosyl residue. However, interaction with the other glucosyl residues is predominantly mediated through water molecules, with the exception of a direct hydrogen bond from N245 to glucosyl residue 3, consistent with the apparent high binding energy at this residue. Hydrophobic interactions with the aromatic sidechain of W358 appear to orient glucosyl residues 2 and 3, while Y341 orients glucosyl residues 4 and 5. In contrast, laminaribiose has its second glucosyl residue positioned to allow direct hydrogen bonding between its O2 and Q176 Oε and O1 and N245. These are the first GH1 glycoside hydrolase family structures to show oligosaccharide binding in the hydrolytic configuration.
KW - Acid-base mutant
KW - Cello-oligosaccharides
KW - Enzyme-substrate complex
KW - Glycoside hydrolase
KW - X-ray crystallography
UR - http://www.scopus.com/inward/record.url?scp=78649906001&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=78649906001&partnerID=8YFLogxK
U2 - 10.1016/j.jsb.2010.09.021
DO - 10.1016/j.jsb.2010.09.021
M3 - Article
C2 - 20884352
AN - SCOPUS:78649906001
VL - 173
SP - 169
EP - 179
JO - Journal of Structural Biology
JF - Journal of Structural Biology
SN - 1047-8477
IS - 1
ER -