The structural basis of oligosaccharide binding by rice BGlu1 beta-glucosidase

Chuenchor, W.; Pengthaisong, Salila; Robinson, Robert; Yuvaniyama, Jirundon; Svasti, Jisnuson; Cairns, James R. Ketudat

doi:10.1016/j.jsb.2010.09.021

Cited by 52 publications

(82 citation statements)

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“…The glycone (Ϫ1) subsite of GH1 glycosidases has been extensively investigated, while less focus has been directed toward the aglycone (ϩ1) subsite, arguably due to the few structures with ligands in the aglycone binding region determined (14). Among the few GH1 structures solved with ligands occupying the aglycone subsites are p-nitrophenyl-␤-thioglucoside in maize Glu1 (Zea mays Glu1 [ZmGlu1]) (15) and cellotetraose in rice ␤-glucosidase (16). In rice, the glycone subsite is formed by direct interactions, while the aglycone subsites mainly consist of water-mediated hydrogen bonds.…”

Section: Resultsmentioning

confidence: 99%

“…The binding site (Fig. 1) is often described as a slot with two walls, where one (here called platform) is highly conserved and the other (here called roof) is more variable (16)(17)(18). Apart from structural data, several mutational studies have been aimed at understanding the substrate specificity of ␤-glycosidases, with a majority focusing on hydrolysis reactions.…”

Section: Resultsmentioning

confidence: 99%

“…The first position chosen for insertion of a phenylalanine was N220, a less conserved position of the ϩ1 subsite. The homologous position in rice ␤-glucosidase, D243, has been shown to participate in water-mediated interactions with short polyglucosides (16). Another interesting position chosen was G222, where mutation to glutamine has resulted in increased transglycosylation (M. Megyeri et al, unpublished data).…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Improved Transferase/Hydrolase Ratio through Rational Design of a Family 1 β-Glucosidase from Thermotoga neapolitana

Lundemo

Adlercreutz

Karlsson

2013

Appl Environ Microbiol

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Alkyl glycosides are attractive surfactants because of their high surface activity and good biodegradability and can be produced from renewable resources. Through enzymatic catalysis, one can obtain well-defined alkyl glycosides, something that is very difficult to do using conventional chemistry. However, there is a need for better enzymes to get a commercially feasible process. A thermostable ␤-glucosidase from the well-studied glycoside hydrolase family 1 from Thermotoga neapolitana, TnBgl1A, was mutated in an attempt to improve its value for synthesis of alkyl glycosides. This was done by rational design using prior knowledge from structural homologues together with a recently generated model of the enzyme in question. Three out of four studied mutations increased the hydrolytic reaction rate in an aqueous environment, while none displayed this property in the presence of an alcohol acceptor. This shows that even if the enzyme resides in a separate aqueous phase, the presence of an organic solvent has a great influence. We could also show that a single amino acid replacement in a less studied part of the aglycone subsite, N220F, improves the specificity for transglycosylation 7-fold and thereby increases the potential yield of alkyl glycoside from 17% to 58%.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Improved Transferase/Hydrolase Ratio through Rational Design of a Family 1 β-Glucosidase from Thermotoga neapolitana

Lundemo

Adlercreutz

Karlsson

2013

Appl Environ Microbiol

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“…The X-ray crystal structures of wild type BGlu1 and of its E176Q acid-base mutant in complexes with oligosaccharides revealed that the glucosyl residues were primarily bound by aromatic-sugar stacking interactions and water-mediated hydrogen bonds with several residues along a long active site cleft, except for the nonreducing terminal glucosyl residue that was distorted through strong hydrogen bonding interactions with the surrounding amino acids. 20,21 To assess the role of the long oligosaccharidebinding cleft of rice BGlu1 E386G glycosynthase in synthesis of long oligosaccharides, its structures alone and in complexes with the donor substrate aglucosyl fluoride or the products cellotetraose and cellopentaose were determined by X-ray crystallography. The importance of the interactions in the long oligosaccharide binding cleft for hydrolysis and glycosynthase production of long cellooligosaccharides was investigated by site-directed mutagenesis of residues in this cleft in the wild type BGlu1 and BGlu1 E386G glycosynthase.…”

Section: Introductionmentioning

confidence: 99%

The role of the oligosaccharide binding cleft of rice BGlu1 in hydrolysis of cellooligosaccharides and in their synthesis by rice BGlu1 glycosynthase

et al. 2012

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Rice BGlu1 b-glucosidase nucleophile mutant E386G is a glycosynthase that can synthesize p-nitrophenyl (pNP)-cellooligosaccharides of up to 11 residues. The X-ray crystal structures of the E386G glycosynthase with and without a-glucosyl fluoride were solved and the a-glucosyl fluoride complex was found to contain an ordered water molecule near the position of the nucleophile of the BGlu1 native structure, which is likely to stabilize the departing fluoride. The structures of E386G glycosynthase in complexes with cellotetraose and cellopentaose confirmed that the side chains of N245, S334, and Y341 interact with glucosyl residues in cellooligosaccharide binding subsites 12, 13, and 14. Mutants in which these residues were replaced in BGlu1 b-glucosidase hydrolyzed cellotetraose and cellopentaose with k cat /K m values similar to those of the wild type enzyme. However, the Y341A, Y341L, and N245V mutants of the E386G glycosynthase synthesize shorter pNPcellooligosaccharides than do the E386G glycosynthase and its S334A mutant, suggesting that Y341 and N245 play important roles in the synthesis of long oligosaccharides. X-ray structural studies revealed that cellotetraose binds to the Y341A mutant of the glycosynthase in a very different, alternative mode not seen in complexes with the E386G glycosynthase, possibly explaining the similar hydrolysis, but poorer synthesis of longer oligosaccharides by Y341 mutants.

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“…Bar=0.1 amino acid substitutions/ site. et al 1995;Burmeister et al 1997;Chuenchor et al 2011;Czjzek et al 2000;Gloster et al 2004;Sanz-Aparicio et al 1998;Verdoucq et al 2004). Although AAGTs show a ten times higher glucosyltransfer activity than glucoside hydrolase activity (Matsuba et al 2010), these two catalytic glutamic acids are nevertheless conserved in AAGTs and in CmAA7GT (Supplementary Figure 3 asterisks).…”

mentioning

confidence: 99%

Isolation of anthocyanin 7-<i>O</i>-glucosyltransferase from Canterbury bells (<i>Campanula medium</i>)

Miyahara

Tani

Takahashi

et al. 2014

Plant Biotechnology

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Canterbury bells (Campanula medium) have deep purple petals due to the accumulation of 7-polyacylated anthocyanin molecules. The first step in the production of 7-polyacylated anthocyanins is glucosylation at the C7 position of anthocyanidin mediated by an acyl-glucose dependent anthocyanin 7-O-glucosyltransferase (AA7GT). To date, two such enzymes have been identified: DgAA7GT from delphinium (Delphinium grandiflorum) and AaAA7GT from African lily (Agapanthus africanus). Here, we describe the isolation of AA7GT cDNA from C. medium and the characterization of the enzymatic properties of a recombinant protein. The CmAA7GT protein belongs to glycoside hydrolase family 1, similarly to other AA7GTs; a phylogenetic analysis revealed that CmAA7GT was in the same clade as other AA7GTs. The CmAA7GT gene showed expression only in flowers, with a peak level of expression at the middle stage of floral development. A recombinant CmAA7GT protein showed significant preference for interaction with anthocyanidin 3-O-rutinoside rather than anthocyanidin 3-O-monoglycoside, which is the preferred target of other AA7GTs. This difference in target preference may reflect a conformational difference in the acceptor pocket of the enzyme protein that recognizes the anthocyanidin glycoside.

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The structural basis of oligosaccharide binding by rice BGlu1 beta-glucosidase

Cited by 52 publications

References 47 publications

Improved Transferase/Hydrolase Ratio through Rational Design of a Family 1 β-Glucosidase from Thermotoga neapolitana

Improved Transferase/Hydrolase Ratio through Rational Design of a Family 1 β-Glucosidase from Thermotoga neapolitana

The role of the oligosaccharide binding cleft of rice BGlu1 in hydrolysis of cellooligosaccharides and in their synthesis by rice BGlu1 glycosynthase

Isolation of anthocyanin 7-<i>O</i>-glucosyltransferase from Canterbury bells (<i>Campanula medium</i>)

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